Phospholipid ether analogs as agents for detecting and locating cancer, and methods thereof

ABSTRACT

The present invention provides methods for treating, detecting and locating recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer selected from the group consisting of Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer and Pancreatic cancer in subject using phospholipid ether analogs.

RELATED APPLICATION

The present application seeks priority from U.S. Provisional ApplicationNo. 60/593,190 filed on Dec. 20, 2004 and is a Continuation-in-Part ofU.S. application Ser. No. 10/906,687 filed on Mar. 2, 2005, which inturn seeks priority from U.S. Provisional Application No. 60/521,166,filed on Mar. 2, 2004, all of which are incorporated herein by referencefor all purposes.

BACKGROUND OF INVENTION

The invention generally relates to phospholipids ether analogs and usethereof and specifically relates to use of phospholipid ether analogsand combinations thereof for diagnosis of metastasis, treatment,pharmacokinetic, dosimetry, and toxicity studies of various cancertypes, such as non-small cell lung cancer, prostate cancer andmetastasis thereof.

Non-Small Cell Lung Cancer (NSCLC)

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deathin the United States today. Surgical resection in appropriately selectedpatients offers the best chance for cure. Accurate pre-operativeassessment of local, regional and distant metastatic spread is thuscritical for optimal management.

Imaging with FDG PET scanning has recently become the “gold standard”for imaging NSCLC, due to improved sensitivity, particularly whencompared with CT imaging. However, its sensitivity for identifyingmediastinal lymph node involvement is only about 90%, and lack ofspecificity; particularly in patients with inflammatory or granulomatousdisease, is particularly problematic. Furthermore, its utility indiagnosing brain tumors or metastases is limited due to high metabolicbackground of normal brain tissue.

Evaluation of the mediastinal lymph node status is essential becausenodal metastasis, which occurs in nearly half of all patients withNSCLC, is probably the most frequent barrier to cure. Accurate stagingmay also spare patients the morbidity of unnecessary, non-curativesurgical procedures. Hence, there remains a need for an imagingtechnique that is more sensitive, specific, and accurate than anycurrently available technology.

Current conventional modalities have limitations. Anatomic imaging withcomputed tomography (CT) and magnetic resonance imaging (MRI) areimpractical for whole body screening but are the most widely usednon-invasive imaging methods for evaluation of loco-regional spread.However, CT relies on size criteria of over one centimeter fordiagnosing abnormal nodes.

Positron-emission tomography (PET) scanning with ¹⁸F-fluorodeoxyglucose(FDG) has generated considerable interest as an oncologic imagingtechnique. A recent study prospectively compared the ability of astandard approach to staging for NSCLC (CT, ultrasound, bone scanning,etc) and PET scanning to detect metastases in mediastinal lymph nodesand distant sites. Mediastinal involvement was confirmedhistopathologically, and distant metastases were confirmed by otherimaging tests. The sensitivity and specificity of PET for detectingmediastinal metastases were 91% and 86%, respectively; for detectingdistant metastases, 82% and 93%, respectively. This compares tosensitivity and specificity for CT scanning of mediastinal involvement75% and 66%, respectively. A meta-analysis involving 39 studies and over1000 patients also found that FDG-PET was more accurate than CT formediastinal staging, (sensitivity and specificity of 85% and 90%,respectively, for FDG-PET and 61% and 79% for CT scanning), althoughFDG-PET became less specific when CT showed enlarged mediastinal lymphnodes (78%).

FDG-PET has been shown to reduce futile thoracotomies in patients.However, because of the false positive and false negative rate,confirmatory mediastinoscopies are often recommended. For example, aretrospective study involving over 200 patients with NSCLC found thatthe sensitivity, specificity, positive and negative predictive values,and accuracy for FDG-PET were 64%, 77%, 45%, 88%, and 75%, respectively.

FDG-PET also plays a role in diagnosing extra-thoracic disease,particularly in patients with intermediate stages of lung cancer. Astudy done by the American College of Physicians involving over 300patients found that unsuspected metastatic disease or second primarymalignancies was identified in 18 of 287 patients (6.3%). Some studies,although not all, suggest that by correctly identifying advanceddisease, PET will avoid unnecessary thoracotomy on 1 in 5 patients.

Conventional anatomic imaging techniques such as CT scanning are alsopoor at predicting survival following treatment. In a recent studyinvolving 73 NSCLC patients receiving treatment with concurrentcisplatin-based chemo/radiotherapy or radiotherapy alone for advanceddisease, response by conventional CT imaging did not correlate withsurvival. Response by FDG-PET scans, however, did correlate stronglywith survival (p<0.001). Survival from the date of a follow-up PET scanwas 84% and 84% at 1 and 2 years respectively for 24 patients who hadachieved a complete response on PET, but only 43% and 31% of the 32patients who did not (p=0.010). These results corroborate similarfindings reported recently by other authors, which also show acorrelation of uptake on PET scan with biological aggressiveness oftumor, and that PET imaging late after completion of treatment is highlypredictive of future survival.

It is generally accepted that FDG-PET imaging is a poor method ofidentifying metastatic disease to the brain in patients with NSCLC.Under normal conditions, the gray matter of the brain has high glucoseutilization and therefore the uptake of FDG is normally high. Whilecerebral metastatic disease is often quite metabolic and often doesdemonstrate increased FDG uptake, it frequently is less than the braingray matter and therefore the cerebral metastases may not beconspicuous. In one series, the sensitivity and specificity for theidentification of cerebral metastatic disease in patients with NSCLC was60% and 99% for FDG-PET, and 100% and 100% for conventional imaging.Therefore, FDG-PET imaging is not considered to be the best method ofevaluating a patient with NSCLC for metastatic disease to the brain.

Another disadvantage of FDG is that it is not specific for tumors, butaccumulates in both malignant and non-malignant hypermetabolic tissues.The overwhelming majority of false positive results (positive resultwhen the radiological abnormality is not due to cancer) with FDG-PETscans of the lung are due to inflammatory and infectious causes. FDG isa nonspecific tracer and accumulates in areas of infection orinflammation. In the lung, these areas can be localized lung parenchymalnodules or more diffuse (subsegmental, segmental or lobar) or in thehilar and mediastinal nodes. In a recent study from Japan, of 116 lungnodules 1-3 cm in diameter, 15 out of 73 malignant nodules were falsenegative on FDG-PET and 15 out of 43 benign nodules were false positiveon FDG-PET. In focal pneumonias causing ground glass opacity nodules,the false positive rate was as high as 80%. In another study, tenpatients with extrapulmonary cancer had false positive FDG-PET uptake inthe lung; 6 had intense focal or multifocal uptake and four had uptakein a more segmental or lobar pattern. In all 10 patients, the uptake wasdue to consolidation or atelectasis and the final diagnosis on follow-upwas pulmonary inflammation or infection. In addition to active bacterialpneumonias, false positive FDG-PET results can occur in many otherinfectious and inflammatory conditions in the lung. In the MidwesternUS, many asymptomatic people have lung nodules and enlarged nodes due toprevious infection with histoplasma; although many of these nodules arequiescent, some represent smoldering or active infection. Pulmonarysarcoidosis is probably one of the more common active inflammatorygranulomatous processes in the lung. Interestingly, when serial FDG-PETscans have been performed in a patient being treated with oralcorticosteroids for pulmonary sarcoidosis, FDG uptake decreased and thenvanished.

FDG-PET is also frequently negative in malignancies with a low metabolicrate, such as bronchoalveolar carcinoma or carcinoid.

Therefore a radiopharmaceutical that could accurately identify earlymetastatic disease in the patients with NSCLC would have a significantimpact on patient care, in terms of both staging and response totherapy. Although PET imaging has improved diagnostic efficacy in thisarea compared to CT, there remains a need for an accurate imagingtechnique that is not based upon metabolic activity, which isnon-specific, but is based upon a tumor-specific function that cannon-invasively screen the whole body, including the brain.

Prostate Cancer

Approximately 230,110 new cases of prostate cancer will be diagnosed inthe United States for the year 2004 alone. Despite technical refinementsin definitive local treatment of clinically organ confined prostatecancer by radical prostatectomy, such that many men are cured withprimary therapy alone, as many as 40% of patients will experiencebiochemical recurrence with long-term follow-up. This recurrence istypically defined as a post-operative PSA level which is greater than orequal to 0.4 ng/ml since patients with PSA levels above this thresholdgenerally develop clinical evidence for recurrence within 6-49 monthsalthough a PSA level of greater than or equal to 0.2 has been proposedmore recently. With limited success, clinical and pathologic criteriaare currently utilized to determine the likelihood for systemic diseaserecurrence. Factors increasing the likelihood of systemic recurrenceinclude a high preoperative PSA level as well as pathologic features ofthe surgical specimen including Gleason score >7, seminal vesicleinvolvement, and lymph node involvement. In contrast, extracapsularextension, positive surgical margins and Gleason score <7 are factorsgenerally associated with local recurrence. In addition, the velocity ofPSA rise following prostatectomy has been utilized to determine whetherdisease recurrence is local or systemic. For instance, Partin et alreported that a PSA rise of less than 0.75 ng/ml/year was morefrequently associated with local recurrence. Furthermore, Patel et alreported that a PSA doubling time of greater than 12 months correlatedwith local recurrence. Despite these clinical and pathologic criteria,the inventors are still unable to accurately select patientsappropriately for local therapy such that many men may receiveunnecessary hormonal ablation.

One of the greatest challenges in treating patients with clinicallyorgan confined prostate cancer or patients with biochemical recurrencefollowing definitive treatment of presumed organ-confined diseaseremains to accurately distinguish localized versus metastatic disease.This diagnostic capability is important to identify patients who maybenefit from effective local treatment modalities including surgery,external beam radiation, brachytherapy, and cryotherapy. Because theinventors presently do not have an accurate means of staging, patientswith occult metastatic disease may unnecessarily undergo local treatmentwith associated risks of therapy. Furthermore, patients with a risingPSA due to local recurrence, in whom systemic recurrence cannot beexcluded with confidence, may unnecessarily undergo hormonal ablation,which is generally not considered curative and is associated withosteoporosis development, decreased libido, weight gain, menopausalsymptoms, and overall malaise, as well as the evolution of hormonallyindependent prostate cancer.

While conventional imaging studies such as computed tomography (CT) andmagnetic resonance imaging (MRI) are useful in assessing soft-tissuemetastasis, the vast majority of prostate cancer metastasizes to thebone only. Thus, the utility of CT and MRI scanning in assessing thedisease is suboptimal and more sensitive imaging modalities for eitherlocally recurrent or metastatic prostate cancer are necessary.Radioimmunoscintigraphy with Indium-111 capromab pendetide (ProstaScint,Cytogen Corp, Princeton, N.J.) has been utilized in patients followingprostatectomy with a rising PSA who have a high clinical suspicion ofoccult metastatic disease and no clear evidence for metastatic diseasein other imaging studies. This scan is based on a radiolabeled murinemonoclonal antibody which is specific for PSMA (Prostate-specificmembrane antigen), a transmembrane protein which is specificallyexpressed by both normal and malignant prostate epithelial cells. WhileProstaScint radioimmunoscintigraphy has been shown to be promising indiagnosing locally recurrent disease in the prostate bed in patientswith rising PSA, clinical results for this scan have been somewhatvariable, with sensitivities ranging between 44% and 92% andspecificities between 36% and 86%. Furthermore, when subsequent biopsywas utilized as the standard of reference for local recurrence,false-negative ProstaScint studies have been reported in 10% to 20% ofcases. In addition, false-positive uptake of ProstaScint has beenreported in neurofibromatosis, lymphomas, renal carcinomas, pelvickidneys, myolipomas, and meningiomas, as well as in the bone marrow ofvertebral bodies. Given this data, use of the ProstaScint scan forpatients at risk for occult metastases from prostate cancer remainscontroversial.

In patients with metastatic prostate cancer, positron-emissiontomography (PET) imaging has recently been used to measure the metabolicactivity of osseous metastases. This technique has proven to beeffective in distinguishing active bony metastasis from osteoblastactivity which occurs as a result of bone healing following successfultreatment of metastatic disease. This question can be assessed better byPET than by either bone scan or CT. Furthermore, changes in PET scanfindings can be seen as early as 4 weeks following initiation ofsystemic treatment in patients with metastatic prostate cancer, whereasin many cases no significant change is seen on conventional bone scan.Therefore imaging utilizing PET technology may be useful in monitoringresponse to treatment in these patients. PET scanning with ¹⁸F-FDG hasgenerated considerable interest as an imaging technique. Recently, ithas been shown that FDG-PET can distinguish between active and quiescentbone metastases in patients with prostate cancer. The intensity of FDGuptake is thought to reflect the metabolic and biological activity ofthese lesions in contrast to the traditional bone scan withtechnetium-diphosphonate compounds in which nonspecific osteoblastactivity may be detected as a false positive signal following treatment.In addition, a false negative reading may be obtained since earlymetastases, which initially seed into the bone marrow, will notnecessarily produce a signal until an osteoblastic response occurs.Therefore, a persistently positive bone scan does not necessarilyindicate the presence of residual viable metastases and a negative bonescan result may not reflect accurately the patient's metastatic tumorburden. FDG-PET may therefore prove beneficial in guiding the managementof patients with bony metastases and in a retrospective study FDG-PETand helical CT have been shown independently to be more effective than¹¹¹In-monoclonal antibody imaging in detecting metastatic disease.

Although the FDG-PET scan is a promising imaging technique in patientswith prostate cancer, most prostate cancers are slow growing andtherefore do not accumulate FDG, and thus do not image well with thatagent. Furthermore, FDG is excreted in the urine and so the accumulationof FDG in the bladder will minimize the probability of detecting localrecurrences of prostate cancer. Indeed Morris et al reported difficultyin detection of soft tissue metastases by FDG-PET alone when metastaticsites are obscured by anatomic pathways of tracer excretion. Morerecently, PET-CT has been found to be more effective than PET alone inidentifying metastatic lesions in patients with suspected occultmetastases. In a prospective study of patients with various tumor types,the specificity and accuracy with multiple radiologic interpretationswere significantly higher for PET-CT.

Accordingly, the need exists for developing a more sensitive andspecific imaging exam, molecular imaging agent, such as phospholipidsether compounds (PLE). It would be desirable to have tumor-selectiveradiopharmaceuticals, with minimal accumulation in the bladder, whichcould accurately identify early metastatic disease in patients withprostate cancer, would have an important impact on patient care, interms of both staging and response to therapy.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting and locatingrecurrence of cancer, radiation and chemo insensitive cancer ormetastasis of cancer selected from the group consisting of Lung cancer,Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovariancancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamouscell cancer, Intestinal cancer, Hepatocellular carcinoma,Retinoblastoma, Cervical cancer, Glioma, Breast cancer and Pancreaticcancer in subject that has or is suspected of having cancer. The methodcomprising the steps of: (a) administering a phospholipid ether analogto the subject; and

(b) determining whether an organ suspected of having recurrence ofcancer, radiation and chemo insensitive cancer or metastasis of cancerin the subject retains a higher level of the analog than surroundingregion(s) wherein a higher retention region indicates detection andlocation of the recurrence of cancer, radiation insensitive and chemocancer or metastasis of cancer.

In a preferred embodiment, the phospholipid analog is selected from:

where X is selected from the group consisting of radioactive isotopes ofhalogen; n is an integer between 8 and 30; and Y is selected from thegroup comprising NH₂, NR₂, and NR₃, wherein R is an alkyl or arylalkylsubstituent or

where X is a radioactive isotope of halogen; n is an integer between 8and 30; Y is selected from the group consisting of H, OH, COOH, COOR andOR, and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. Further, in certainembodiments, X is selected from the group of radioactive halogenisotopes consisting of ¹⁸F, ³⁶Cl, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I and ²¹¹At. More preferably, the phospholipid ether is18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope. In this method,preferably the detection is carried out by a of PET, CT, MRI scanningmethods combination thereof.

Another embodiment of the present invention provides a method for thetreatment of recurrence of cancer, radiation and chemo insensitivecancer or metastasis of cancer in a subject. The method comprisesadministering to the subject an effective amount of a compoundcomprising a phospholipid ether analog. In a preferred embodiment, therecurrence of cancer, radiation and chemo insensitive cancer ormetastasis of cancer occurs in the group selected from Lung cancer,Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovariancancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamouscell cancer, Intestinal cancer, Hepatocellular carcinoma,Retinoblastoma, Cervical cancer, Glioma, Breast cancer, Pancreaticcancer and Carcinosarcoma. Also preferably, the phospholipid analog isselected from:

where X is selected from the group consisting of radioactive isotopes ofhalogen; n is an integer between 8 and 30; and Y is selected from thegroup comprising NH₂, NR₂, and NR₃, wherein R is an alkyl or arylalkylsubstituent or

where X is a radioactive isotope of halogen; n is an integer between 8and 30; Y is selected from the group consisting of H, OH, COOH, COOR andOR, and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. In this method,preferably, X is selected from the group of radioactive halogen isotopesconsisting of ¹⁸F, ³⁸Cl, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,²¹¹At and combinations thereof. More preferably, the effective amount ofphospholipid ether analog is a combination of at least two isotopes, onewith one with a path range of about 0.1 Å to 1 mm and a second with apath range of about 1 mm to 1 m.

Most preferably, the effective amount of phospholipid ether analog is acombination of at least two isotopes, ¹²⁵I and ¹³¹I. Also, preferably,the phospholipid ether is 18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[1,8-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.

In certain embodiments, the effective amount of phospholipid etheranalog is fractionated. In yet other embodiments, the effective amountof phospholipid ether analog is about 0.5 μCi to about 3 Ci treatable ina linear and dose dependent manner. Other embodiments provide that thedosage is adaptable to the cancer-volume. Yet other embodiments providethat the dosage for radiation insensitive tumor is greater than dosagefor radiation sensitive tumor and less than 3 Ci and is adaptable to thecancer-volume.

Another embodiment of the present invention provides the use ofphospholipid ether analog for the production of a pharmaceuticalcomposition for the treatment of recurrence of cancer, radiation andchemo insensitive cancer or metastasis of cancer. In this embodiment,the phospholipid analog is selected from:

where X is selected from the group consisting of radioactive isotopes ofhalogen; n is an integer between 8 and 30; and Y is selected from thegroup comprising NH₂, NR₂, and NR₃, wherein R is an alkyl or arylalkylsubstituent or

where X is a radioactive isotope of halogen; n is an integer between 8and 30; Y is selected from the group consisting of H, OH, COOH, COOR andOR, and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. Also in this embodiment,preferably X is selected from the group of radioactive halogen isotopesconsisting of ¹⁸F, ³⁶Cl, ⁷⁸Br, ⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,²¹¹At and combinations thereof. Also preferably, the phospholipid etheris 18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.

Further objects, features and advantages of the invention will beapparent from the following detailed description, drawings and appendedclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Structures of certain PLE analogs

FIG. 2. Classical models showing the production of bremsstrahlung,characteristic X-rays, and Auger electrons. (left) Electrons arescattered elastically and inelastically by the positively chargednucleus. The inelastically scattered electron loses energy, whichappears as bremsstrahlung. Elastically scattered electrons (whichinclude backscattered electrons) are generally scattered through largerangles than are inelastically scattered electrons. (right) An incidentelectron ionizes the sample atom by ejecting an electron from aninner-shell (the K shell, in this case). De-excitation, in turn,produces characteristic X-radiation (above) or an Auger electron(below). Secondary electrons are ejected with low energy from outerloosely bound electron shells, a process not shown.

FIG. 3. Scintigraphy of the anterior chest of Patient 03 acquired at 1,2, and 6 days after iv administration of 1 mCi 131I-NM-324. Uptake isseen in the left lingular lung cancer (T) with increasingtumor-to-background ratios over time.

FIG. 4. Time course (days) of NM404 in a SCID mouse with a human RL-251adrenal tumor (T) xenograft.

FIG. 5. Tissue distribution I-1125-NM404 in RL 251 Adrenal Cancer inSCID mice depicting that while accumulation in the tumor increased,distribution in blood, spleen and kidney reduced by days 1 through 14.

FIG. 6. Apparent SCC1 and SCC6 tumor regression after injection of125I-NM404. By day 41, the tumor is significantly reduced.

FIG. 7. The image shows one of the tumor-bearing animals treated with250 μCi of I-125-NM404 at 4 weeks following injection. The hair abovethe tumor has fallen off, presumably due to the strong accumulation ofradioactivity in the tumor. Additionally, the surface of the tumorsappears “caved in” and shows darker areas, presumably from hemorrhageand necrosis. The figure shows the effect of I-125-NM404 on the tumor.Although tumor size (outer dimensions) may not shrink, I-125-NM404causes central necrosis. The measurement method measuring outer tumordimensions may have underestimated tumor volume response followingI-125-NM404.

FIG. 8. PC3 human prostate cancer model implanted into SCID mouse. PC3is known to be radiation insensitive. The curves between control (noradioactivity, cold NM404) and treatment (I-125-NM404) only separateabout 4-5 weeks after treatment, until then the growth of the tumorslooks the same indicating that: 1) NM404 takes a few days to about 1week to fully accumulate in the tumor, and 2) the isotope I-125 has alow radiation flow (since it has a long half life). Both factorscontribute to a delayed onset of the therapy effect, at a time when allNM404 has cleared out of normal tissues.

FIG. 9. Scintigraphic comparison of NM404 (bottom panel) and NM324 (toppanel) at 1, 2, and 4 days in a SCID mouse with human prostate PC-3tumor (arrow) implanted in the flank. Liver and background radioactivityare much improved with NM404.

FIG. 10. Tumor volume for each group was recorded over the 10-weekassessment period depicting the control and dosage of 50, 150, 250 and500 μCi. In this figure, control animals show rapidly growing tumorsover the 10-week assessment period. This confirms that the compounditself C-NM404 has no substantial effect on tumors growth. The 50 μCidose group did not show any difference to control animals, hence theseseem to be ineffective dose levels in this animal model. However, the150, 250 and 500 μCi dose groups show a substantial and prolongedtreatment effect. Tumor volumes are stable and same tumors appear“collapsed” (the tumor surface has caved in). Additionally, hair abovethe tumors fell off confirming substantial accumulation of radioactivityin these tumors. The results show a dose-linear effect of I-125-NM404 ontumor volume.

FIG. 11. A549 Tumor Xenografts (1×10⁶ cells, s.c.) in Female SCID MiceFractionated Dose (3×50 mCi), having 2 independent controls for eachdosage. A fractionated dosing of NM404 (e.g. 3×50 micro-Ci versus asingle dose of 150 micro-Ci) produced the same therapy effect. Thefractionated dose may be safer since it is eliminated from normaltissues in between fractionated injections.

FIG. 12. A549 Large Tumors vs Small Tumors 150 microcuries

FIG. 13. Bioscan image (A) obtained 4 days post ¹²⁵I-NM404 injectioninto an Apc^(Min) mouse. Digital photo (B) and positionally matchedfused image (C) of excised mouse lung containing spontaneous lung tumor(2 mm dia, arrow) showing intense uptake of NM404 into the tumor.

FIG. 14. Time course of ¹²⁵I-NM404 in human RL-251 adrenal cancerxenograft in a SCID mouse. Prolonged tumor (1.5×0.5 cm, arrow) retentionis evident even after 20 days.

FIG. 15. Fused 3D surface-rendered MRI image (blue) and 3D microPETimage (A) obtained 24 h after iv injection of ¹²⁴I-NM404 (80 μCi) into arat with a CNS-1 glioma brain tumor. Images were fused using Amira(v3.1). Lower panels show (B) contrast-enhanced coronal MRI slicethrough the tumor (arrow) and (C) fused coronal MRI and ¹²⁴I-NM404microPET images corroborating presence and location of the tumor.

FIG. 16. ¹²⁴I-MicroPET images a human A549 lung tumor xenograft in aSCID mouse 48 h post injection of ¹²⁴I-NM404 (80 μCi). Correspondingimaging planes are indicated by dashed green line in coronal view.

FIG. 17. ¹²⁴I-MicroPET images (coronal, sagittal, and axial) 96 h afteriv injection of ¹²⁴I-NM404 into a PC-3 (flank) tumor-bearing mouse. Nobladder activity was evident at any time point

FIG. 18. ¹²⁴I-MicroPET images of a transgenic mouse with a spontaneousc-myc pancreatic adenocarcinoma (5 mm) 18 h post injection of ¹²⁴I-NM404(80 μCi).

FIG. 19. Scintigraphic comparison of NM404 (bottom panel) and NM324 (toppanel) at 1, 2, and 4 days in a SCID mouse with human prostate PC-3tumor (arrow) implanted in the flank. Liver and background radioactivityare much improved with NM404.

FIG. 20. Time course of ¹²⁵I-NM404 in human RL-251 adrenal cancerxenograft in a SCID mouse. Prolonged tumor (1.5×0.5 cm, arrow) retentionis evident even after 20 days.

FIG. 21. Photo of excised prostate/vesicular gland complex (A). Bioscanimage obtained 4 days post NM404 injection (B). Positionally matchedfused photo/Bioscan image of excised prostate/vesicular gland (C)showing intense uptake of radioactivity in the prostate tumor.

FIG. 22. High density 3D surface-rendered microCT images of a Nude mouseleg at various times following intratibial injection (arrow in firstpanel depicts injection site and direction) of 2×10⁵ human PC3 prostatetumor cells. Tumor begins protruding through the bone by 28 days (arrow)and by 46 days the tumor has literally destroyed the tibia leaving onlythe fibula intact.

FIG. 23. Co-registered surface-rendered and coronal slice microCT imagefused with positionally-matched Bioscan radionuclide scan (color) 4 daysfollowing injection of ¹²⁵I-labeled NM404. Focal NM404 activitycorrelates well with tibial PC3 tumor (arrow).

FIG. 24. Fused 3D surface-rendered MRI image (blue) and 3D microPETimage (A) obtained 24 h after iv injection of ¹²⁴I-NM404 (100 μCi) intoa rat with a CNS-1 glioma brain tumor. Images were fused using Amira(v3.1). Right panels show (B) contrast-enhanced coronal MRI slicethrough the tumor (arrow) and (C) fused coronal MRI and ²⁴¹I-NM404microPET images corroborating presence and location of the tumor.

FIG. 25. Posterior whole body planar nuclear medicine image (A) 4 daysafter iv administration of ¹³¹I-NM-404 (0.8 mCi) to a patient with nonsmall cell lung cancer (6 cm dia., arrow). Lung tumor is easily detectedin corresponding axial (B) and coronal (C) computed tomography (CT)scans.

I. GENERAL DESCRIPTION OF THE INVENTION

General Description of the Invention: Before the present methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, and reagents described,as these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the chemicals, cell lines, vectors, animals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

As defined herein, the term “isomer” includes, but is not limited tooptical isomers and analogs, structural isomers and analogs,conformational isomers and analogs, and the like. In one embodiment,this invention encompasses the use of different optical isomers of ananti-tumor compound of Formula 3A. It will be appreciated by thoseskilled in the art that the anti-tumor compounds useful in the presentinvention may contain at least one chiral center. Accordingly, thecompounds used in the methods of the present invention may exist in, andbe isolated in, optically-active or racemic forms. Some compounds mayalso exhibit polymorphism.

It is to be understood that the present invention may encompass the useof any racemic, optically-active, polymorphic, or stereroisomeric form,or mixtures thereof, which form possesses properties useful in thetreatment of tumor-related conditions described and claimed herein. Inone embodiment, the anti-tumor compounds may include pure (R)-isomers.In another embodiment, the anti-tumor compounds may include pure(S)-isomers. In another embodiment, the compounds may include a mixtureof the (R) and the (5) isomers. In another embodiment, the compounds mayinclude a racemic mixture comprising both (R) and (S) isomers. It iswell known in the art how to prepare optically-active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase).

The invention includes the use of pharmaceutically acceptable salts ofamino-substituted compounds with organic and inorganic acids, forexample, citric acid and hydrochloric acid. The invention also includesN-oxides of the amino substituents of the compounds described herein.Pharmaceutically acceptable salts can also be prepared from the phenoliccompounds by treatment with inorganic bases, for example, sodiumhydroxide. Also, esters of the phenolic compounds can be made withaliphatic and aromatic carboxylic acids, for example, acetic acid andbenzoic acid esters. As used herein, the term “pharmaceuticallyacceptable salt” refers to a compound formulated from a base compoundwhich achieves substantially the same pharmaceutical effect as the basecompound.

This invention further includes method utilizing derivatives of theanti-tumor compounds. The term “derivatives” includes but is not limitedto ether derivatives, acid derivatives, amide derivatives, esterderivatives and the like. In addition, this invention further includesmethods utilizing hydrates of the anti-tumor compounds. The term“hydrate” includes but is not limited to hemihydrate, monohydrate,dihydrate, trihydrate and the like.

This invention further includes methods of utilizing metabolites of theanti-tumor compounds. The term “metabolite” means any substance producedfrom another substance by metabolism or a metabolic process.

As defined herein, “contacting” means that the anti-tumor compound usedin the present invention is introduced into a sample containing thereceptor in a test tube, flask, tissue culture, chip, array, plate,microplate, capillary, or the like, and incubated at a temperature andtime sufficient to permit binding of the anti-tumor compound to areceptor. Methods for contacting the samples with the anti-tumorcompound or other specific binding components are known to those skilledin the art and may be selected depending on the type of assay protocolto be run. Incubation methods are also standard and are known to thoseskilled in the art.

In another embodiment, the term “contacting” means that the anti-tumorcompound used in the present invention is introduced into a patientreceiving treatment, and the compound is allowed to come in contact invivo.

As used herein, the term “treating” includes preventative as well asdisorder remittent treatment. As used herein, the terms “reducing”,“suppressing” and “inhibiting” have their commonly understood meaning oflessening or decreasing. As used herein, the term “progression” meansincreasing in scope or severity, advancing, growing or becoming worse.As used herein, the term “recurrence” means the return of a diseaseafter a remission.

As used herein, the term “administering” refers to bringing a patient,tissue, organ or cells in contact with an anti-tumor phospholipid ethercompound. As used herein, administration can be accomplished in vitro,i.e. in a test tube, or in vivo, i.e. in cells or tissues of livingorganisms, for example, humans. In certain embodiments, the presentinvention encompasses administering the compounds useful in the presentinvention to a patient or subject. A “patient” or “subject”, usedequivalently herein, refers to a mammal, preferably a human, thateither: (1) has a disorder remediable or treatable by administration ofthe anti-tumor substance using a phospholipid ether compound or (2) issusceptible to a disorder that is preventable by administering theanti-tumor compound using a phospholipid ether compound

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the anti-tumor compound having radioactivitytogether with suitable diluents, preservatives, solubilizers,emulsifiers, and adjuvants, collectively “pharmaceutically-acceptablecarriers.” As used herein, the terms “effective amount” and“therapeutically effective amount” refer to the quantity of activetherapeutic agent sufficient to yield a desired therapeutic responsewithout undue adverse side effects such as toxicity, irritation, orallergic response. The specific “effective amount” will, obviously, varywith such factors as the particular condition being treated, thephysical condition of the patient, the type of animal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of thecompounds or its derivatives. In this case, an amount would be deemedtherapeutically effective if it resulted in one or more of thefollowing: (a) the prevention of disease (e.g., pancreatic cancer,breast cancer); and (b) the reversal or stabilization of such disease.The optimum effective amounts can be readily determined by one ofordinary skill in the art using routine experimentation.

Pharmaceutical compositions are liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween (Polysorbate) 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils).

Also encompassed by the invention are methods of administeringparticulate compositions coated with polymers (e.g., poloxamers orpoloxamines). Other embodiments of the compositions incorporateparticulate forms protective coatings, protease inhibitors or permeationenhancers for various routes of administration, including topical,parenteral, pulmonary, nasal and oral. In one embodiment thepharmaceutical composition is administered parenterally, paracancerally,transmucosally, tansdermally, intramuscularly, intravenously,intradermally, subcutaneously, intraperitonealy, intraventricularly,intracranially and intratumorally.

Further, as used herein “pharmaceutically acceptable carriers” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.9% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's and fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

Controlled or sustained release compositions administerable according tothe invention include formulation in lipophilic depots (e.g. fattyacids, waxes, oils). Also comprehended by the invention are particulatecompositions coated with polymers (e.g. poloxamers or poloxamines) andthe compound coupled to antibodies directed against tissue-specificreceptors, ligands or antigens or coupled to ligands of tissue-specificreceptors.

Other embodiments of the compositions administered according to theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,including parenteral, pulmonary, nasal and oral.

Compounds modified by the covalent attachment of water-soluble polymerssuch as polyethylene glycol, copolymers of polyethylene glycol andpolypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified compounds (Abuchowski et al., 1981;Newmark et al., 1982; and Katre et al., 1987). Such modifications mayalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. As a result, the desired in vivo biological activity may beachieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In yet another method according to the invention, a pharmaceuticalcomposition can be delivered in a controlled release system. Forexample, the agent may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity to the therapeutic target, for example liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990).

The pharmaceutical preparation can comprise the anti-tumor compoundalone, or can further include a pharmaceutically acceptable carrier, andcan be in solid or liquid form such as tablets, powders, capsules,pellets, solutions, suspensions, elixirs, emulsions, gels, creams, orsuppositories, including rectal and urethral suppositories.Pharmaceutically acceptable carriers include gums, starches, sugars,cellulosic materials, and mixtures thereof. The pharmaceuticalpreparation containing the anti-tumor compound can be administered to apatient by, for example, subcutaneous implantation of a pellet. In afurther embodiment, a pellet provides for controlled release ofanti-tumor compound over a period of time. The preparation can also beadministered by intravenous, intra-arterial, or intramuscular injectionof a liquid preparation oral administration of a liquid or solidpreparation, or by topical application. Administration can also beaccomplished by use of a rectal suppository or a urethral suppository.

The pharmaceutical preparations administerable by the invention can beprepared by known dissolving, mixing, granulating, or tablet-formingprocesses. For oral administration, the anti-tumor compounds or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are mixed with additives customary for this purpose, suchas vehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions. Examples of suitable inert vehicles are conventional tabletbases such as lactose, sucrose, or cornstarch in combination withbinders such as acacia, cornstarch, gelatin, with disintegrating agentssuch as cornstarch, potato starch, alginic acid, or with a lubricantsuch as stearic acid or magnesium stearate.

Examples of suitable oily vehicles or solvents are vegetable or animaloils such as sunflower oil or fish-liver oil. Preparations can beeffected both as dry and as wet granules. For parenteral administration(subcutaneous, intravenous, intra-arterial, or intramuscular injection),the anti-tumor compounds or their physiologically tolerated derivativessuch as salts, esters, N-oxides, and the like are converted into asolution, suspension, or expulsion, if desired with the substancescustomary and suitable for this purpose, for example, solubilizers orother auxiliaries. Examples are sterile liquids such as water and oils,with or without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solutions, and glycols such as propylene glycols or polyethyleneglycol are preferred liquid carriers, particularly for injectablesolutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Such compositions may beprepared as aerosols delivered to the nasopharynx or as injectables,either as liquid solutions or suspensions; however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like or any combination thereof.

In addition, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentswhich enhance the effectiveness of the active ingredient.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts, which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the anti-tumor compounds or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another method according to the invention, the active compound can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353-365 (1989); Lopez-Berestein ibid., pp. 317-327; seegenerally ibid).

For use in medicine, the salts of the anti-tumor compound may bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts of the compounds include acid addition salts which may,for example, be formed by mixing a solution of the compound according tothe invention with a solution of a pharmaceutically acceptable acid suchas hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaricacid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalicacid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

Generally, phospholipids ether compounds and specially NM404 is apromising new tumor-selective diagnostic imaging agent to monitor thetreatment response of several tumor treatment modalities. RadioiodinatedNM404, a second-generation phospholipid ether analog, had displayedremarkable tumor selectivity in 27/27 tumor models. Due to a lack ofmetabolic phospholipase enzymes in the membranes of tumor cells, theprevailing hypothesis of this approach is that phospholipid etheranalogs become trapped exclusively in tumor cell membranes because oftheir inability to become metabolized and eliminated. Thus, thedifferential clearance rates of phospholipid ethers from normal cellsversus viable tumor cells form the basis of this concept. Resultsobtained in a variety tumor models indicate that NM404 is sequesteredand selectively retained by viable tumor cells and localizes in bothprimary and metastatic lesions regardless of anatomic location includingthose found in lymph nodes. Unlike FDG, this agent does not localize ininfectious sites. Other advantages of NM404 over FDG include thefollowing: NM404 is selective for and retained indefinitely by malignanttumor cells whereas FDG in not selective for tumor cells and goes toinfectious sites and hyperplasias (Barret's Esophagus). Further, since¹²⁴I has a 4 day physical half life it can be shipped anywhere in theworld whereas FDG with its 110 min half-life, may have limiteddistribution within 200 miles of the production site. NM404 undergoesprolonged retention (not metabolized) and therefore affords asignificant therapeutic potential when mated with an appropriateradioisotope like ¹³¹I whereas FDG does not possess any therapeuticpotential. NM404 can be labeled with a variety of iodine isotopesexpanding it versatility (diagnosis and therapy as well as a tool forexperimental animal studies) whereas FDG is limited to ¹⁸F for PETscanning or potentially ¹⁹F (stable) for magnetic resonance imagingalbeit at very low sensitivity levels. Regardless of its tumor targetingability, due to its rapid metabolism in tumor cells, it has notpotential for therapy. NM404 affords the potential to not onlyaccurately predict local tumor response to various treatment modalities,but also allows detection of distant metastatic lesions in cases ofsub-therapeutic primary tumor treatment.

The PLE compounds may be designed to more accurately estimate thespecificity and sensitivity of radiolabeled PLE analogs such as NM404 asan imaging agent in prostate cancer and other cancers. Based uponpreclinical models, PLE analogs such as NM404 are likely to exhibit highuptake in tumors giving this agent the significant potential for use instaging, following response to therapy, or potentially as a therapeuticagent when coupled with higher doses of ¹³¹I, ¹²⁵I, or ²¹¹At analpha-emitting halogen with therapeutic efficacy.

II. PREFERRED EMBODIMENTS

The present invention generally provides methods and techniques for thedetection and treatment of various cancers. The present inventionprovides a method for detecting and locating recurrence of cancer,radiation and chemo insensitive cancer or metastasis of cancer selectedfrom the group consisting of Lung cancer, Adrenal cancer, Melanoma,Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Livercancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer,Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma,Breast cancer and Pancreatic cancer in subject that has or is suspectedof having cancer. The method comprising the steps of: (a) administeringa phospholipid ether analog to the subject; and

(b) determining whether an organ suspected of having recurrence ofcancer, radiation insensitive cancer or metastasis of cancer in thesubject retains a higher level of the analog than surrounding region(s)wherein a higher retention region indicates detection and location ofthe recurrence of cancer, radiation and chemo insensitive cancer ormetastasis of cancer.

In a preferred embodiment, the phospholipid analog is selected from:

where X is selected from the group consisting of radioactive halogenisotopes; n is an integer between 8 and 30; and Y is selected from thegroup comprising NH₂, NR₂, and NR₃, wherein R is an alkyl or arylalkylsubstituent or

where X is a radioactive halogen isotope; n is an integer between 8 and30; Y is selected from the group consisting of H, OH, COOH, COOR and OR,and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. Further, in certainembodiments, X is selected from the group of radioactive halogenisotopes consisting of ¹⁸F, ³⁶Cl, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I and ²¹¹At. More preferably, the phospholipid ether is18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[1,8-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope. In this method,preferably the detection is carried out by PET CT MRI scanning methodsand combination thereof.

As used herein “alkyl” group refers to a straight chain, branched orcyclic, saturated or unsaturated aliphatic hydrocarbons. The alkyl grouphas 1-16 carbons, and may be unsubstituted or substituted by one or moregroups selected from halogen, hydroxy, alkoxy carbonyl, amido,alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino,carboxyl, thio and thioalkyl. A “hydroxy” group refers to an OH group.An “alkoxy” group refers to an —O-alkyl group wherein alkyl is asdefined above. A “thio” group refers to an —SH group. A “thioalkyl”group refers to an —SR group wherein R is alkyl as defined above. An“amino” group refers to an —NH₂ group. An “alkylamino” group refers toan —NHR group wherein R is alkyl is as defined above. A “dialkylamino”group refers to an —NRR′ group wherein R and R′ are all as definedabove. An “amido” group refers to an —CONH₂. An “alkylamido” grouprefers to an —CONHR group wherein R is alkyl is as defined above. A“dialkylamido” group refers to an —CONRR′ group wherein R and R′ arealkyl as defined above. A “nitro” group refers to an NO₂ group. A“carboxyl” group refers to a COOH group.

As used herein, “aryl” includes both carbocyclic and heterocyclicaromatic rings, both monocyclic and fused polycyclic, where the aromaticrings can be 5- or 6-membered rings. Representative monocyclic arylgroups include, but are not limited to, phenyl, furanyl, pyrrolyl,thienyl, pyridinyl, pyrimidinyl, oxazolyl, isoxazolyl, pyrazolyl,imidazolyl, thiazolyl, isothiazolyl and the like. Fused polycyclic arylgroups are those aromatic groups that include a 5- or 6-memberedaromatic or heteroaromatic ring as one or more rings in a fused ringsystem. Representative fused polycyclic aryl groups include naphthalene,anthracene, indolizine, indole, isoindole, benzofuran, benzothiophene,indazole, benzimidazole, benzthiazole, purine, quinoline, isoquinoline,cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine,pteridine, carbazole, acridine, phenazine, phenothiazine, phenoxazine,and azulene. Further, as used herein “arylalkyl” refers to moieties,such as benzyl, wherein an aromatic is linked to an alkyl group which islinked to the indicated position in the PLE compound.

Another embodiment of the present invention provides a method for thetreatment of recurrence of cancer, radiation and chemo insensitivecancer or metastasis of cancer in a subject. The method comprisesadministering to the subject an effective amount of a compoundcomprising a phospholipid ether analog. In a preferred embodiment, therecurrence of cancer, radiation and chemo insensitive cancer ormetastasis of cancer occurs in the group selected from Lung cancer,Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovariancancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamouscell cancer, Intestinal cancer, Hepatocellular carcinoma,Retinoblastoma, Cervical cancer, Glioma, Breast cancer, Pancreaticcancer and Carcinosarcoma. Also preferably, the phospholipid analog isselected from:

where X is selected from the group consisting of radioactive halogenisotopes halogen; n is an integer between 8 and 30; and Y is selectedfrom the group comprising NH₂, NR₂, and NR₃, wherein R is an alkyl orarylalkyl substituent or

where X is a radioactive halogen isotope; n is an integer between 8 and30; Y is selected from the group consisting of H, OH, COOH, COOR and OR,and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. In this method,preferably, X is selected from the group of radioactive halogen isotopesconsisting of ¹⁸F, ³⁶Cl, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,²¹¹At and combinations thereof. More preferably, the effective amount ofphospholipid ether analog is a combination of at least two isotopes, onewith a path range of about 0.1 Å to 1 mm and a second with a path rangeof about 1 mm to 1 m, also as discussed in FIG. 2.

Most preferably, the effective amount of phospholipid ether analog is acombination of at least two isotopes, ¹²⁵I and ¹³¹I. Also, preferably,the phospholipid ether is 18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.

As shown in FIG. 11 in certain embodiments, the effective amount ofphospholipid ether analog is fractionated. The advantage of usingfractionated dosage is that it allows for the PLE analog to be removedfrom normal tissues. As an example, fractionated dosing of NM404 (e.g.3×50 micro-Ci versus a single dose of 150 micro-Ci) produced the sametherapy effect, while still providing low doses of compounds which isthen eliminated from normal tissues in between fractionated injections.

In yet other embodiments, the effective amount of phospholipid etheranalog is about 0.5 μCi to about 500 mCi, and as shown in FIG. 10, thisis treatable in a linear and dose dependent manner. Other embodimentsprovide that the dosage is adaptable to the cancer-volume as shown inFIG. 12. The graph in FIG. 12 shows the difference in tumor growth forthe same dose of I-125-NM404 (150 microCi) when injecting in animalswith small (<1 cm) and large tumors (>1 cm).

The results show that a small tumor showed stunned tumor growth withthat dose, however a large tumor population did not show any effects forthe same dose, basically behaving like non-radiated control. The graphin FIG. 12 further illustrates is that the effective tumor dose shouldbe adjusted to tumor volume and that there is a tumor dose per volume oftumor volume that has to be achieved in order to show efficacy.

Yet other embodiments provide that the dosage for radiation and chemoinsensitive tumor is greater than dosage for radiation sensitive tumorand less than 500 mCi and is adaptable to the cancer-volume, asascertainable by comparing PC3 (FIG. 8) and A549 cancer models (FIGS.10, 11 and 12), in which PC3 model is radiation insensitive.

Another embodiment of the present invention provides the use ofphospholipid ether analog for the production of a pharmaceuticalcomposition for the treatment of recurrence of cancer, radiation andchemo insensitive cancer or metastasis of cancer. In this embodiment,the phospholipid analog is selected from:

where X is selected from the group consisting of radioactive halogenisotopes; n is an integer between 8 and 30; and Y is selected from thegroup comprising NH₂, NR₂, and NR₃, wherein R is an alkyl or arylalkylsubstituent or

where X is a radioactive halogen isotope; n is an integer between 8 and30; Y is selected from the group consisting of H, OH, COOH, COOR and OR,and Z is selected from the group consisting of NH₂, NR₂, and NR₃,wherein R is an alkyl or arylalkyl substituent. Also in this embodiment,preferably X is selected from the group of radioactive halogen isotopesconsisting of ¹⁸F, ³⁶Cl, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²², ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,²¹¹At and combinations thereof. Also preferably, the phospholipid etheris 18-(p-Iodophenyl)octadecyl phosphocholine,1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine,wherein iodine is in the form of a radioactive isotope.

Synthesis and Structure Activity Relationship Effects on the TumorAvidity of Radioiodinated Phospholipid Ether Analogs

Radioiodinated phospholipid ether analogs have shown a remarkableability to selectively accumulate in a variety of human and animaltumors in xenograft and spontaneous tumor rodent models. It is believedthat this tumor avidity arises as a consequence of metabolic differencesbetween tumor and corresponding normal tissues. The results of thisstudy indicate that one factor in the tumor retention of these compoundsin tumors is the length of the alkyl chain that determines theirhydrophobic properties. Decreasing the chain length from C12 to C7resulted in little or no tumor accumulation and rapid clearance of thecompound in tumor-bearing rats within 24 hours of administration.Increasing the chain length had the opposite effect, with the C15 andC18 analogs displaying delayed plasma clearance and enhanced tumoruptake and retention in tumor-bearing rats. Tumor uptake displayed bypropanediol analogs NM-412 and NM-413 was accompanied by high levels ofliver and abdominal radioactivity 24 hours post injection to tumorbearing rats. Addition of a 2-O-methyl moiety to the propanediolbackbone also retarded tumor uptake significantly. A direct comparisonbetween NM-404 and its predecessor, NM-324, in human PC-3 tumor bearingimmune-compromised mice, revealed a dramatic enhancement in both tumoruptake and total body elimination of NM-404 relative to NM-324. Based onimaging and tissue distribution studies in several rodent tumor models,the C18 analog, NM-404, was chosen for follow-up evaluation in humanlung cancer patients. Preliminary results have been extremely promisingin that selective uptake and retention of the agent in tumors isaccompanied by rapid clearance of background radioactivity from normaltissues, especially those in the abdomen. These results strongly suggestthat extension of the human trials to include other cancers iswarranted, especially when NM-404 is radiolabeled with iodine-124, a newcommercially available positron-emitting isotope. The relatively longphysical half-life of 4 days afforded by this isotope appears wellsuited to the pharmacodynamic profile of NM-404.

Chemical Synthesis.

In the course of synthesis of iodinated phospholipid ether analogs theinventors sought to develop a general synthetic scheme which would allowaltering the chain length in the target compounds. The syntheticapproach was based on the copper catalyzed cross-coupling reaction ofGrignard reagents with alkyl tosylates or halides¹⁵ (Schemes 1, 2). Thechoice of the building blocks for alkyl chain elongation was dictated bythe commercial availability of starting materials.

The synthesis was initiated by the conversion of p-iodobenzyl alcohol 11to p-iodobenzyl bromide 12 as shown in Scheme 1. p-Iodobenzyl bromidewas further coupled with Grignard reagent derived from THP protected11-bromoundecanol 13 in the presence of 0.5-0.7 mol % of Li₂CuCl₄.12-(p-Iodophenyl)dodecanol 17 obtained after deprotection of the firstcoupling product 16 was used for the synthesis of C12 iodinatedphospholipids as described earlier.^(12,13) Alcohol 17 also served as astarting material for the synthesis of ω-iodophenyl alkanols with longerchains. For example, coupling of the tosylate 18 with Grignard reagentsmade from bromides 14 and 15 followed by THP deprotection furnished theC15 (20) and C18 (22) alcohols, respectively. These alcohols wereconverted into corresponding alkylphosphocholines 5 (NM-397) and 6(NM-404) according to published procedures.^(12,13) Propanediolphospholipid ethers 7 (NM-413) and 8 (NM-412) were synthesized from3-benzyloxypropanol 25, and 2-O-methyl-rac-glycerol phospholipid ethers9 (NM-414) and 10 (NM-410) were obtained from1-O-benzyl-2-O-methyl-rac-glycerol 26 using a sequence of reactions theinventors had previously reported. Radiolabeling with iodine-125 wasaccomplished by an isotope exchange method routinely employed in thelaboratory.

Biology.

The avidity of PLE analogs to localize in tumors was evaluated inseveral animal models. The PC-3 model represents a human tumor cell linethat was used to determine the target (tumor) to non-target ratio ofNM404 and NM412 in head to head comparison in order to select acandidate for an initial human pharmacokinetic trial in prostate cancerpatients. The MatLyLu (Dunning R3327 rat) model, a rat prostate tumorline, was used specifically to screen 9 specific analogs prior toentering them into control for dosimetry and tumor-bearing animals fordetermining tumor/background ratios. Finally, the Walker-256carcinosarcoma model was used for quantitative tissue distributionpurposes.

In order to expedite the screening process and minimize the number oftumor bearing animals utilized in multiple time point tissuedistribution studies, new radioiodinated homologs were imaged by gammacamera scintigraphy in the rat Dunning R3337 (MAT LyLu strain) prostatecancer model. Thus, male Copenhagen rats received subcutaneous injectionof MAT LyLu cells (1×10⁶ cells) in the thigh 10-14 days prior toinjection of the radioiodinated PLE analogs (30-40 μCi) in 2% Tween-20solution. Gamma camera images were obtained at multiple time pointsincluding 24 and 48 hours post injection. Homologs (NM-410, NM-413, andNM-414) displaying high hepatic uptake, significant abdominalaccumulation and retention or poor tumor uptake and retention were notsubmitted to subsequent biodistribution analysis. Tissue distribution ofradioactivity in rats bearing Walker-256 carcinosarcoma was assessed atvarious time intervals following intravenous administration of theradioiodinated chain length homologs. The first group of compounds thatwas tested included three alkylphosphocholines: a shorter chain analogwith seven carbons 3 (NM-396) and two analogs with a longer chainlength, 5 (NM-397, C15 alkyl chain length) and 6 (NM-404, C18 alkylchain length).

Initial biodistribution experiments performed with 3 (NM-396, C7 analog)indicated rapid tissue clearance accompanied by significant in vivodeiodination. By 24 hours, the amount of radioactivity in the thyroidwas 213% ID/g, whereas levels of radioactivity in all of the organssurveyed was <0.10% ID/g. As follows from these studies, reducing thenumber of methylene groups to seven apparently afforded a much morehydrophilic molecule which was rapidly excreted by the kidneys. Incontrast to compound 4 (NM-346, the C12 analog), the C7 analog 3 clearedrapidly from the rat and did not localize in tumor tissue at any of thetime points examined.

This observation directed the future efforts to assessing the effect ofincreasing the length of the aliphatic chain upon tumor uptake andretention. The tissue distribution of the C15 homolog 5 (NM-397) wasassessed utilizing the same Walker 256 rat tumor model. Radioactivity inthe tumor increased with time and peaked at 48 hours afteradministration (1.65±0.23% of ID/g) as opposed to most normal tissueswhich exhibited their highest levels of radioactivity 6 hours afteradministration. With the exception of thyroid, the tumor had higherradioactivity concentrations at 24, 48, and 120 hours than any of theother tissues surveyed. A more rapid washout of radioactivity occurredin the normal tissues as compared to the tumor presumably due tometabolism and elimination by normal tissues. The accumulation ofradioactivity in the thyroid increased throughout the course of thestudy suggesting the presence of a low level of in vivo deiodination.Levels of radioactivity in the duodenum were similar to those of tumorwith maximum levels being observed at 48 hours after administration(1.38±0.24% ID/g).

Although limited results were obtained with the C12 analog 4 (NM-346) inthis model, results suggest that the tissue distribution profile of theC15 analog 5 (NM-397) was similar to that observed with the C12 analog 4with the exception of a 2-fold increase in tumor uptake at 24 h.Remaining uptake and clearance in other organs and tissues was similarbetween the two compounds.

The effect of further extending the aliphatic chain to the C18 analog 6(NM-404) depicted a dynamic profile of this compound that was similar tothe C15 analog 5 (NM-397) as levels of radioactivity peaked in the tumor48 hours after administration (1.14±0.01% of ID/g), albeit at slightlylower levels. Quantities of radioactivity detected in liver, kidney andduodenum were significantly lower following administration of the C18compound 6 as compared to the same organs in the C15 analog 5 studies.In addition, the C18 analog 6 was retained in the circulation to a muchgreater extent than the other chain length homologs surveyed. Forexample, at 120 hours, blood levels for 6 were 0.6±0.1% of ID/g ascompared to levels of 0.07±0.00% of ID/g for the C15 (5) analog. Totalradioactivity levels in the thyroid were relatively low in both 5 and 6when the extremely small mass of the gland is considered.

In order to examine the transport properties of PLE analogs, plasma wasisolated from Walker-256 tumor-bearing rats 7 days after administrationof iodine-125 labeled 6. The distribution of radioactivity in the plasmacompartment of a rat receiving 6 (NM-404) was studied. PAGE analysisrevealed that most of the circulating radioactivity (88%) was associatedwith the albumin fraction following administration of the C18 analog 6.This finding is similar to results reported by Eibl who studied bindingof phospholipid ether prototype, ET-18-OCH₃, with serum proteins andfound that the majority of the ether lipid (71%) was bound to albuminand about 6% to HDL.

Comparative Imaging Studies.

Gamma camera scintigraphy images shown in FIG. 19 directly compare thetumor uptake and body clearance of second generation analog ¹²⁵I-NM-404(6) versus its shorter chain, first generation predecessor, ¹²⁵I-NM-324(2) at 1, 2, and 4 days post administration in immune-compromised SCIDmice bearing human PC-3 prostate tumor xenografts. Qualitativescintigraphic comparison of these two PLE analogs demonstrated astriking difference in tumor uptake and overall body clearance. Thelonger chain agent, NM-404, displays rapid tumor uptake and prolongedretention accompanied by rapid whole body elimination of radioactivity,whereas tumor uptake and body clearance are substantially delayed withNM-324, even at 4 days following administration. Significant tumoruptake and retention of C18 analog NM-404 accompanied by rapid wholebody elimination clearly defined the superior imaging properties ofNM-404 in this model.

Extensive quantitative tissue distribution results obtained at 1, 3, 5,and 8 days following administration of radioiodinated NM-404 in thismodel indicated rapid elimination of radioactivity from all normaltissues over the 8 day evaluation period. Tumor uptake, however,continued to increase up until day 5 when it reached 18% injected doseper gram of tumor. Tumor to background tissue ratios steadily increasedover the course of the experiment due to prolonged retention in tumorscoupled with a steady elimination from normal tissues. Tumor tobackground tissue ratios exceeded 4, 6.8, 23, and 9 in blood, liver,muscle, and prostate, respectively, 3 days after injection and continuedto improve at 5 and 8 days. Again, although thyroid levels ranged from26 to 54% injected dose per gram of tissue, these levels are actuallyquite low and represent an extremely small percentage of the injecteddose when the exceedingly small mass of the organ is considered and thedata are presented on a percent administered dose per organ basis.

Kötting et al have investigated the effects of alkyl chain length on thebiodistribution of three alkyl phosphocholine (APC) analogs. The Köttingstudy differed from the experiments in that 1) compounds were orallyadministered at concentrations thought to be cytotoxic to tumor, and 2)the C22 compound contained a double bond in the alkyl chain. Thereforedirect comparisons with the work described herein cannot be made due tolarge dose differences and the unknown bioavailability of the oralagents. In the Kötting study, C16, C18 and C22 analogs were administeredorally to rats bearing a methylnitrosourea-induced mammary carcinoma indaily doses of 50-120 ∝mol/kg. As alkyl chain length increased, theobserved levels of compound in kidney, liver, and lung decreased. Incontrast to the tracer results obtained with the radioiodinated PLEanalogs, Kötting and coworkers found that levels of APC in blooddecreased with increasing chain length, while tumor levels increasedwith increasing chain length.¹⁹ It would be expected that oraladministration would result in a substantial amount of degradation ofthe ether lipids in the GI tract prior to absorption.

Tumors were readily visualized with the C12 (4), C15 (5) and C18 (6)alkyl phosphocholine homologs via gamma camera scintigraphy at both 24and 48 hours after injection. Rat imaging results obtained with C15 (7,NM-413) and C18 (8, NM-412) propanediol analogs, on the other hand,displayed tumor uptake accompanied by high liver and abdominalradioactivity levels. Imaging results obtained with C15 (9, NM-414) andC18 (10, NM-410) 2-O-Me glycerol analogs in the MAT LyLu prostate modelindicated high radioactivity levels in the liver and abdomen with littleto no uptake of the agent into tumors. The differences in the clearanceand quantity of radioactivity from non-target tissues including theliver and intestinal tract will have significant impact upon theapplication of radioiodinated phospholipid ether analogs as imagingagents in humans. Non-target tissue uptake can decrease the efficacy ofradiodiagnostic imaging by creating high background activity or bycausing excessive exposure of radiosensitive tissues to the injectedradioactivity. A preliminary clinical trial with 2 (NM-324, themeta-iodo isomer of NM-346) in cancer patients, while affordingexcellent tumor uptake, was limited by the radiation dosimetryassociated with accumulation in non-target tissues including liver,kidneys, and bladder.

The strategy was to examine the alkyl portion of phospholipid etheranalog structure and determine its role in tumor localization andretention. Qualitative rat whole body screening scans acquired inMAT-LyLu tumor bearing rats with radioiodinated PLE analogs with longerchain lengths revealed sufficient tumor uptake to permit detection.However, follow-up tissue distribution studies have shown thatsequential increases in the chain length from C12 to C15 to C18,resulted in a rapid decline in the amount of radioactivity detected inthe non-target organs. This substantial decrease in non-target tissueactivity was accompanied by a relatively small reduction in the levelsof radioactivity present in the tumor. In addition, the C18 analog 6(NM-404) displayed a propensity to remain in the circulation much longerthan the C12 (4) and C15 (5) analogs. A longer plasma half-life may beexpected to result in additional opportunities for uptake of the C18compound 6 by the tumor as it continually circulated through thevasculature. This extended plasma half-life may be a result of strongbinding of the probe to albumin. Uptake and transport of radiolabeledPLE by plasma components may also be an important factor related to thetumor retention of these compounds. Certainly, increase of the chainlength from C7 to C18, results in an increase in the lipophilicity ofthe PLE analogs. Greater lipophilicity may increase the affinity ofthese compounds for the cell membrane, and may alter their binding toplasma components. Uptake and transport in the circulation by endogenouslipoproteins such as LDL and HDL may also impact the biologicaldistribution into the tumor.

In preparation for human clinical trials, unlabeled NM404 was subjectedto independent (University of Buffalo Toxicology Research Center) acutetoxicity evaluation at 1200 times the anticipated imaging mass dose inrats and rabbits. The agent was well tolerated and no acute toxicitieswere found at this dose level. Due to its selective tumor uptake andretention properties in a variety of rodent tumor models and subsequentexcellent safety profile in rats and rabbits, NM-404 was selected toundergo initial human pharmacokinetic evaluation in non-small cell lungcancer (NSCLC) patients. Patients underwent planar gamma-camerascintigraphy after receiving an injection of ¹³¹I-NM-404 (<1 mCi).Preliminary human results (n=3) demonstrated tumor uptake and prolongedretention in the primary lung tumors (FIG. 25). Relative to the highliver uptake values observed with its predecessor, NM-324, however,liver and abdominal radioactivity levels were much lower with NM-404,suggesting the feasibility of evaluating this agent in other abdominaltumors including those associated with the colon, prostate, andpancreas.

Conclusions.

In summary, the rationale behind the design of new iodinated PLE analogsdescribed in this paper was exploited in an effort to evaluate theeffect of chain length and other structural modifications in thehydrophobic part of the phospholipid molecule on tumor uptake andretention. Decreasing the chain length from C12 to C7 resulted in littleor no tumor accumulation and rapid clearance of the compound intumor-bearing rats by 24 hours after administration. Increasing thechain length had the opposite effect, with the C15 and C18 analogsdisplaying delayed plasma clearance and enhanced tumor uptake andretention in tumor-bearing rats. Tumor uptake displayed by propanediolanalogs 7 and 8 was accompanied by fairly high levels of liver andabdominal radioactivity 24 hours post injection to tumor bearing rats.Addition of a 2-O-methyl moiety in 9 and 10 retarded tumor uptakesignificantly. A direct comparison between NM-404 and its predecessor,NM-324, in human PC-3 bearing immune-compromised mice, revealed adramatic enhancement in both tumor uptake and total body elimination ofNM-404 relative to NM-324. NM-404 afforded superior imaging propertiesto the other analogs examined in several animal models, thus warrantingfurther evaluation of this second generation PLE analog in human lungcancer patients. Preliminary clinical results in humans indicateddesired tumor uptake and retention properties similar to those seenpreviously in animal models. In contrast to its shorter chainpredecessor NM-324, however, NM-404 displayed significantly lower liver,kidney and abdominal background radioactivity levels, which in additionto providing promise for lung tumor imaging, suggests further evaluationof this agent in human colorectal, pancreatic and prostate cancerpatients is warranted. Moreover, a lack of urinary bladderradioactivity, suggests little renal clearance of the agent or itsmetabolites over the time points examined. This represents a significantadvantage over 18F-fluorodeoxyglucose (FDG), a PET agent used routinelyfor tumor imaging today, which undergoes significant renal elimination,thus prohibiting imaging in the area of the prostate.

Because the tumor-targeting strategy of PLE analogs appears to involveselective tumor retention over time, relatively short-lived nuclidessuch as ¹⁸F or even ^(99m)Tc are not practical for labeling NM-404 atthe current time. Given the preliminary success of ¹³¹I-NM-404 in thecurrent lung cancer imaging trial, it is now imperative to radiolabelNM-404 with iodine-124, a relatively new positron-emitting isotope witha 4.2-day half-life, and to evaluate its tumor detection efficacy byPET. It has been reported that PET imaging with ¹²⁴I affords over 40times the sensitivity of planar ¹³¹I-gamma imaging. PET, unliketraditional gamma-camera imaging, also offers significant resolutionenhancement and 3-dimensional imaging capabilities, as well as superiorquantitation properties relative to planar scintigraphic imaging. Thelong, 4-day physical half-life of this PET isotope is well suited to thetumor uptake and retention kinetics of NM-404 and the inventors are inthe process of extending the imaging studies with NM-404 to include PET.

Non-Small Cell Lung Cancer (NSCLC)

Phospholipid ether analogs (PLE) molecules have unique biochemical andpharmacological properties resulting in a high degree of tumorselectivity. Unlike FDG, which accumulates non-specifically in bothmalignant and non-malignant hypermetabolic tissues, the inventors haveshown radioiodinated PLE analogs undergo selective retention in a widevariety of murine and human tumors in high levels, and do not accumulatein normal or inflammatory tissues. The family of phospholipids ethers(PLE) are characterized by the presence of an ether-linked long chainalkyl or alkenyl alcohol connected to a glycerophosphocholine moleculeordinarily found in mammalian cells as a minor component of the totalphospholipid content. Among the PLE, there are many subtypes, one of themost extensively studied subtypes are structurally simplified alkylphosphocholines (APC). I-125-NM404, is part of this APC family.

The present invention teaches methodologies for preliminary human PETimaging data regarding the use of the second-generation PLE analog,NM404, in imaging patients with NSCLC. In preclinical models, theinventors have shown that NM404 is (a) selectively retained in 27/27tumor models, including lung and brain tumors as well as PC-3 prostatebone metastases, (b) is not retained in normal, premalignant, orhyperplastic tissues and, (c) is not retained in inflammatory tissues.In first-in-human pharmacokinetic studies with ¹³¹I-NM404 in patientswith NSCLC, the inventors have found that ¹³¹I-NM404 is safe, and thatfrom 24-48 hours is the optimal scintigraphic imaging time point fortumor detection. These studies also revealed significantly lower liverand background activity levels relative to earlier promising analogs andconfirmed the agent doesn't cross the intact blood brain barrier.

Although sufficient for early human pharmacokinetic studies, the poorimaging characteristics and planar imaging nature associated withiodine-131 scintigraphy dictate that NM404 be further developed for PETimaging. The inventors have recently radioiodinated NM404 in excellentyield with commercial iodine-124, a relatively new, long-lived PETisotope, the half-life of which appears ideally matched to thepharmacokinetic profile of NM404. Initial microPET scans obtained with¹²⁴I-NM404 in xenograft and spontaneous mouse and rat tumor modelsconfirmed universal tumor avidity and prolonged retention. Extendingthese results to PET scanning is now necessary in order to accuratelycharacterize and quantitate the in vivo distribution properties of thisagent. The primary objective of this proposal is to further developNM404 for PET/CT imaging in NSCLC patients with this radioisotope.

The present invention studies (a) the efficacy of imaging primary NSCLCtumors with ¹²⁴I-NM404 PET/CT in patients with NSCLC undergoingresection, by comparing pre-operative images with pathological findings,(b) the specific tumor accumulation and metabolic fate of NM404 in NSCLCpatients undergoing resection, and correlate tumor retention withdecreased phospholipase-D activity; (c) preliminary data regarding thesensitivity of imaging locoregional, and metastatic tumors with¹²⁴I-NM404 PET/CT in patients with NSCLC, by comparing these resultswith FDG PET/CT scanning; and (d) preliminary data regarding thespecificity of imaging with ¹²⁴I-NM404 PET/CT, by imaging patients whopresent with solitary pulmonary nodules, or have a diagnosis ofpulmonary sarcoidosis or granulomatous infections such as fungal ormycobacterial infections, or bacterial pneumonias.

Due to its prolonged tumor retention properties, ¹²⁵I-labeled NM404 hasrecently afforded significant tumor regression (vide infra) in SCID micebearing A549 human lung tumor xenografts. Exhibiting both diagnostic andtherapeutic utility, NM404 is being developed as a true, potentiallyuniversal, diagnostic and therapeutic agent.

NM404 has now been evaluated in 27 animal tumor models, includingseveral lung, and it is clear that once the agent enters tumor cells, itreaches a metabolic dead end and becomes trapped. Prolonged tumorretention of this agent is demonstrated in a human adrenal tumorxenograft implanted into SCID mice (FIG. 14). NM404 is also retained inspontaneous murine lung tumors (FIG. 13). Using ¹²⁵I-labeled NM404, theinventors have been able to image mammary and prostate tumors in mice inexcess of 60 days. Prolonged tumor retention characteristics maysignificantly enhance the radiotherapeutic efficacy of the agent.Imaging and tissue distribution studies performed in mouse models aimedat determining the uptake characteristics in a wide variety of tumormodels are summarized in Table 2.

Rationale for Isotope Selection for Clinical Studies.

Although the inventors have studied NM404 labeled with iodine-131 in thepreliminary human pharmacokinetic studies, this is less than optimal forimaging since iodine-131 scintigraphy produces images with limitedresolution and little anatomic detail. The new long-lived isotopeiodine-124, however, will produce tomographic PET images which will bedisplayed with corresponding CT images, thereby providing significantlygreater anatomic and functional detail.

Because the tumor-targeting strategy of PLE analogs appears to involveselective tumor retention over time, relatively short-lived nuclidessuch as ¹⁸F or even ^(99m)Tc are not practical for labeling NM404 at thecurrent time. Although the use of other isotopes, including iodine-123,may ultimately prove suitable for use with NM404 in certain tumors, thecurrent focus will develop the PET imaging capability of this agent dueto the recent success of oncologic imaging using hybrid PET-CT scanners.The unsurpassed diagnostic accuracy afforded by a biochemical orfunctional tumor imaging PET agent combined with the precise anatomicaccuracy provided by CT is now the gold standard for tumor imaging.However, it is highly advantageous to label PLE analogs with iodine-124,a relatively new PET isotope, wherein the physical half-life (4 days)matches well with PLE tumor uptake and retention kinetics. LabelingNM404 with iodine-124 represents a natural extension of the currentstudies with gamma-emitting nuclides due to its 4-day physicalhalf-life. It has been shown that PET imaging with ¹²⁴I affords over 40times the sensitivity of planer ¹³¹I-gamma scintigraphy. PET, unliketraditional gamma camera imaging, not only offers significant resolutionenhancement and 3-dimensional capabilities, but when used in conjunctionwith hybrid PET-CT also affords exquisite image quantitation due tobuilt in attenuation correction benefits afforded by CT. Due to thepreliminary success of ¹³¹I-NM404 in the current lung cancer imagingtrial, it is now imperative to evaluate the tumor detection efficacy ofNM404 labeled with iodine-124 by PET in order to overcome thelimitations inherently associated with planer scintigraphy.

Successful Radiolabeling of NM404 with iodine-124

The inventors have obtained high specific activity sodium-Iodide-124 in0.1 N NaOH from Eastern Isotopes (Sterling, Va.). Radiolabeling of NM404is achieved in greater than 60% isolated radiochemical yield bymodification of an isotope exchange method. Briefly, a 2-ml glass vialis charged with 10 mg of ammonium sulfate dissolved in 50 μl ofdeionized water. Glass beads are added, a Teflon lined septum and screwcap are added and the vial gently swirled. A solution of 10 μg (in 10 μlof ethanol) of stock NM404 is added followed by aqueous sodiumiodide-124 (1-5 mCi) in less than 30 μl aqueous 0.01 N sodium hydroxide.The reaction vile is swirled gently. A 5-ml disposable syringecontaining glass wool in tandem with another 5-ml charcoal nugget filledsyringe with needle outlet are attached. The glass wool syringe acts asa condensation chamber to catch evaporating solvents and the charcoalsyringe traps free iodide/iodine. The reaction vessel is heated in aheating block apparatus for 45 minutes at 150° C. after which Four 20 mlvolumes of air are injected into the reaction vial with a 25-mldisposable syringe and allowed to vent through the dual trap attachment.The temperature is raised to 160° C. and the reaction vial is heatedanother 30 minutes. After cooling to room temperature, ethanol (200 μl)is added and the vial swirled. The ethanolic solution is passed througha pre-equilibrated Amberlite IRA 400-OH resin column to remove unreactediodide. The eluent volume is reduced to 50 μl via a nitrogen stream (usecharcoal syringe trap) and the remaining volume injected onto a silicagel column (Perkin Elmer, 3 μm×3 cm disposable cartridge column elutedat 1 ml/min with hexane/isopropanol/water (52:40:8)) for purification.Final purity is determined by TLC (plastic backed silica gel-60 elutedwith chloroform-methanol-water (65:35:4, Rf=0.1). The HPLC solvents areremoved by rotary evaporation and the resulting radioiodinated NM404 issolubilized in aqueous 2% Polysorbate-20 and passed through a 0.22 μmfilter into a sterile vial. Radiochemical purity is typically greaterthan 99%.

In-Vivo PET Imaging of Murine Tumors with ¹²⁴I-NM404.

Brain Tumors.

The inventors evaluated the imaging characteristics of NM404 in C6glioma (3-5 mm in diameter) tumor-bearing and sham-operated rats. Tissuedistribution analysis was performed with ¹²⁵I-NM404 at 24 and 48 hoursafter injection, and a separate group of animals subjected to microPETscanning at various times following injection of ¹²⁴I-NM404.Biodistribution analysis indicated minimal NM404 radioactivity in normalbrain tissue, however, tumor/brain ratios (% injected dose/g) were 10.6,and 12.0 at 24, 48 hours, respectively. (FIG. 15) NM404 tumor uptake wascorroborated by histology. These preliminary results obtained in a ratglioma model suggest that NM404 holds considerable promise for thedetection of malignant primary and metastatic brain tumors.

Lung, Prostate, and Pancreatic Tumor Models

Preliminary microPET imaging results with ¹²⁴I-NM404 in lung, prostateand pancreatic mouse models are presented in FIGS. 16-18. A commonfeature in all microPET images obtained in animal models is the lack ofbladder activity at any time point. Human pharmacokinetic studies haveconfirmed this finding as only 4% of the agent is cleared renally within4 days of iv injection (majority is excreted via GI route). In allcases, NM404 displayed significant tumor avidity regardless of locationin the body. Tumor uptake was typically scene within 6 hours ofinjection, albeit tumor to background generally improved significantlyover time, especially in abdominal tumors.

Stimulated by these and other observations, the inventors recentlyconducted a small ¹²⁵I-NM404 pilot therapy study in SCID mice with humanA549 lung tumor xenografts. ¹²⁵I-NM404 was administered as either asingle dose or alternatively in 3 doses (once a week for 3 weeks) togroups of 6 mice and a separate cohort received an equivalent mass doseof unlabeled NM404 for comparison. Single doses were 50 or 500 μCi andthe repeat dose group received a total of 3 weekly 50 μCi doses. Tumorgrowth was monitored for 10 weeks following final injection. Preliminaryresults indicate a significant regression in tumor growth at the higherdose and perhaps a similar response in the 3-dose group, although thesearms of the study are still ongoing. New arms are now being initiated tocover the dose levels between the current ones in order to moreaccurately evaluate the therapeutic potential of this agent. Even at the500 μCi dose none of the mice showed any signs of toxicity.

Clinical Trials:

NM404 has been administered in a tracer dose for imaging (0.3 μg/kg bodymass) in the Phase 1 NSCLC trial at the University of Wisconsin. A 70 kgsubject would thus receive approximately 21 μg of NM404, although recentimprovements in the exchange labeling procedure have resulted in muchsmaller mass doses being injected. Given the recent improvements inlabeling and specific activity, the inventors were able to already reacha mass dose in the range of 142.9 ng/kg BW which corresponds to 0.22nmol/kg BW or a total mass dose of 0.010 mg per 70 kg patient. Furtherimprovements are anticipated based on a new labeling methodology asdescribed below, which will likely result in a greater than 50-foldreduction in the mass of compound required. Taken those futureimprovements into account, inventors believe that the intended clinicalmass dose of I-125-NM404 to be given in the clinical trials performedwould be roughly 3-5 ng/kg BW or about 250 ng per 70 kg patient.

The subset of phospholipid ethers known as alkyl phosphocholinespossesses a wide range of pharmacologic activities, having been studiedextensively as anticancer and anti-leshmanial agents at micromolarconcentrations in animal models and in humans. Neither the mode ofuptake nor the precise mechanism(s) of action have been clearly defined,although disruption of membrane lipid metabolism is observed in tumorcell membranes. Miltefosine, hexadecyl phosphocholine, has a reportedLD50 of 606 nmol/kg in rats with a maximum tolerated dose of 39 nmol/kgover a 4-week period. In human clinical trials, a daily oral dose of 150mg (3×50 mg) was well tolerated with minimal side effects (nausea andemesis) (Planting A S, Stoter G, Verweij J. European Journal of Cancer1993; 29A: 518-519).

Toxicology Studies Performed for NM404

This section will summarize four formal GLP toxicology studies performedusing NM404. These studies are:

Report/ Observation Study No. Administration Animals Period GLP Study 27Single Dose Male Rats 14 Days Yes Study 28 Single Dose Male Rabbits 14Days Yes Study 31 Single Dose Female Rats 14 Days Yes Study 32 SingleDose Female Rabbits 14 Days Yes

Formal toxicology studies of NM404 in male rats and rabbits wereperformed at the Toxicology Research Center of the State University ofNew York at Buffalo under the direction of Dr. Paul Kostyniak. Drugvehicle and drug product were provided to Dr. Kostyniak by Dr. RaymondCounsell of the University of Michigan for testing as described in theappended synopses of Study 27 and Study 28. Since no significant toxiceffects were noted at a dose of 4 mg/kg, which is a dose approximately200 times the anticipated imaging dose at that time, and the inventorsestimate to be approximately 2860 times the anticipated therapy dose forclinical trials under this invention. Human safety studies of unlabeledNM404 were initiated in normal male humans at a mass of 10 times theanticipated imaging dose and about 21 times the anticipated therapydose. The results again showed no toxicity attributable to the drugsubstance. This toxicology study was performed under GLP conditions.

Subsequently, the researchers at the University of Wisconsin initiated atoxicology study of unlabeled NM404 in female rats and rabbits (Study 31and Study 32) at the Toxicology Research Center at SUNY-Buffalo in orderto expand the patient population to be studied in the NSCLC Phase 1clinical trial. Since no significant toxic effects were noted at a doseof 0.04 mg/kg, which is a dose approximately 200 times the revisedimaging mass dose at that time, and what the inventors estimate to beapproximately 286 times the anticipated therapy dose for clinicaltrials. Human safety studies of unlabeled NM404 were initiated in normalfemale humans at a mass of 10 times the initially anticipated imagingdose and about 21 times the anticipated therapy dose. Again, nosignificant toxic effects were noted in either female rats or rabbits.This toxicology study was performed under GLP conditions.

The toxicology studies were planned at that time to go up to 200 timesthe anticipated mass dose in human trials. Since then, improvements tothe exchange labeling methodology (see Method 2 in the CMC section) willlikely result in a greater than 50-fold reduction in the mass ofcompound required for the reaction. Thus, recalculation of thetoxicology dose indicated that no toxicity was evident at a dose of atleast 10,000× the anticipated clinical dose. During the Phase 1 safety,pharmacokinetic and dosimetry trials in normal volunteers and NSCLCpatients, no toxicity has been observed in either the normal male (U MI)or female (UW) volunteers or in any of the NSCLC patients participatingin the study (UW).

Control and Test Articles:

The test article was a solution of C-NM404 (active ingredient)containing inactive ingredients (vehicle). The control solution for thisstudy was the vehicle without the active ingredient.

The test article was formulated as follows:

(1) Active Ingredient: 2 mg/mL C-NM404 (2) Inactive Ingredient: 2%Polysorbate20 in sterile water (injection grade)

The control was formulated as follows:

(1) Inactive Ingredient: 2% Polysorbate20 in sterile water (injectiongrade)

The control and test articles were received from Raymond E. Counsell,Ph.D., Professor of Pharmacology & Medicinal Chemistry, Department ofPharmacology, The University of Michigan Medical School, Ann Arbor,Mich. on Oct. 29, 1998. At the Toxicology Research Center at theUniversity of Buffalo, the study test site, the four (4) vials of testarticles labeled “NM-404 (2 mg/ml) in 2% Polysorbate 20/Sterile Water,MAL-V1-82” and four (4) vials of control articles labeled “ControlVehicle-2% Polysorbate 20 in Sterile Water, MAL-V1-83” were inventoriedand stored at room temperature.

Administration:

The test article (C-NM-404) was administered at greater than 200 timesthe anticipated clinical dose. The control and test rats were injectedintravenously in the lateral tail vein. The rats were injected with thetest or control article intravenously at 2 ml/kg of body weight using a25 gauge needle and a 1-ml syringe. The injections were given byalternating rats from the control group with rats from the test group.The injections were given cautiously over a 30 second to one minute timeinterval. The injection to control rat #27-01 was given at 9:03 am andlast injection to test rat #27-16 was given at 11:01 am. The followingrats moved during the injection and received multiple injections:control #6 (2 sites), test #9 (2 sites), test #15 (3 sites).

In human applications, ET-18-OCH3, a true PLE and thus more dissimilarto NM404 than miltefosine, (edelfosine, mouse LD50 (oral) 200 mg/kg) isadministered intravenously at a dose of 15-20 mg/kg/d at 5 mg/ml in 5%HSA. The maximum tolerated dose is 50 mg/kg. Adverse effects reportedfor this agent include pulmonary edema impaired hepatic function andhemolysis for up to 4 hours post-injection. (Berdel W E, Fink U,Rastetter J. Lipids 1987; 22:967-969). Given that the total mass dose ofNM404 would be less than one ten thousandth of the daily individual doseof Miltefosine, no toxic events would be anticipated.

Study Procedures:

The rats were observed by LAF personnel for signs of acute toxicity fromthe time of injection until 3:30 μm. The rats were weighed five (5)times a week (Monday through Friday) and their body weights, recorded inkilograms. On Dec. 17, 1998 the rats were anesthetized with sodiumpentobarbital (65 mg/ml, Lot#970789, Expiration Date: Feb. 1, 2000)administered intraperitoneally. A heart puncture was then performedusing a 20 gauge needle and a 10 ml syringe to collect the blood samplesfor the hematological CBC and the clinical blood chemistries. The ratswere exsanguinated to cause death. The thymus, heart, lungs, spleen,kidneys, liver, brain, and testes were collected, examined grossly,weighed and sectioned for pathology. The tissue samples (except thymus)were placed in jars of “Z-fix” fixative for histology.

Sixteen (16) Sprague-Dawley rats were received from Harlan-SpragueDawley, Indianapolis, Ind. All rats were males born on the same date andappeared to be healthy. They were housed at the Laboratory AnimalFacility and given food and water ad lib. Each rat was given a two-partnumber starting with 27 (the study number) and followed by a ‘unique’number of ‘01’ to ‘16’ (numerical). Each rat was ear punched in theright ear with the unique number of ‘01’ to ‘16’. The control rats werenumbered from #27-01 to #27-08 and the test rats were numbered from#27-09 to #27-16. The unique numbers were also applied to each cageindicating which rats were housed within that cage. There were fourcages of control rats and four cages of test rats. The rats were weighedand the mean weight of the control group was 0.234 kilograms and 0.238kilograms for the test group. The two groups of eight (8) rats wereestablished by random assignment. The rats were weighed daily until thetermination of the study on Day 14. Test product, dose and mode ofadministration, batch number: C-NM-404 (MAL-V1-82) 2 mg/ml, given viatail vein injection over 30-60 seconds Duration of Treatment: Singledose Reference therapy, dose and mode of administration, batch number:2% Polysorbate20 in sterile water, 2 ml/kg of body weight, given viatail vein injection over 30-60 seconds.

Safety: On Day 14, the blood samples for both groups will be analyzedfor hematology and clinical chemistry. Additionally, the followingorgans will be removed for pathology report and histology slides: brain,lung, liver, heart, kidney, spleen, and testes. A compilation chart oforgan weights with organ/weight ratios for each rat may be compiled.

Statistical Methods:

Differences in body weights and biochemical parameters will be comparedbetween groups using t-test.

Safety Results:

No unusual behavior was noted in any of the rats during this time orthroughout this 14 day study. The tail vein injection sites were checkeddaily when the rats were weighed and no adverse tissue reactions werenoted in any of the rats. The mean weights of the rats in control groupand the test group were not significantly different, despiteinsignificant periodic day-to-day weight loss in both groups.

The gross examination of tissues was performed one week followingsacrifice by a pathologist, at SUNY at Buffalo. No gross lesions werenoted in the test or control group. The tissue sections were thenanalyzed by light microscopic examination. There were no changes in thehistopathology of the organs examined that can be attributed to theadministration of the test material. Rat #27-12 receiving the testmaterial has a small, focal area of myocardial injury (infarct in anearly stage). This change is not seen in the other section of hear thatwas also processed so is interpreted as being quite small. Since thelesion is not seen in other animals receiving the material, thepathologist felt it is attributable to some unexplained alteration. Itis not due to infection arising from the lung since the lungs do notshow histopathological change.

Results of clinical blood chemistries and hematology were checked forany obvious values that were above or below the reference range. Thesevalues resulted in a group comparison using t-test at a p-value of 0.05.T-tests were performed on: phosphorus, sodium, potassium, AST, ALT,alkaline phosphatase, globulin, A/G ratio, glucose, WBC, RBC andhemoglobin. There were no significant differences found.

CONCLUSION: No acute toxicologic effects attributable to the testarticle had been found. There were no significant differences betweenthe test and control group.

Similar tests were performed on male and female rabbits and female ratsand no adverse effects attributable to the test article were noted.

Preclinical Pharmacology:

Inventors approach to the development of safe and effective cancertherapies is to design small-molecule carrier molecules which arecapable of being selectively retained in cancer tissue, but not orminimally in non-cancerous tissues. An extension of this approach toradiotherapy would exploit the selective delivery of theradiopharmaceutical to deposit therapeutic levels of radiation withinthe tumor mass while minimizing radiation-induced damage to normaltissues. This technology is based on the unique biochemical andpharmacological properties of phospholipid ethers (PLE's) and especiallyits sub-group alkyl phosphocholine analogues, such as NM404, whichdisplays a high degree of tumor selectivity. Phospholipids are anessential component of cellular membranes where they impart structuralintegrity and are intimately associated with a variety of cell signalingprocesses. Phosphatidylcholine, commonly known as lecithin, is such anexample. Phospholipid ethers, on the other hand, represent a minorsubclass of phospholipids that also reside in membranes. As the nameimplies, these lipids contain an ether rather than an ester linkage atthe C-1 position. Platelet-activating factor (PAF) represents one of thebetter known phospholipid ethers.

Based on his early findings that several animal and human tumorscontained much higher concentrations of naturally occurring ether lipidsin their cell membranes than normal tissue (Snyder, F. and Wood R.Cancer Res. 1968; 28:972-978, Snyder F. and Wood R. Cancer Res. 1969;29:251-258), Snyder proposed that the accumulation of ether lipids intumors arose as a result of a lower capacity of tumor cells tometabolize these lipids. The prevailing hypothesis is that phospholipidethers become trapped in tumor membranes because of their inability tobecome metabolized and eliminated, likely by a phospholipase enzyme inthe cell membrane. This hypothesis is supported by experiments showingthat lipid extraction of tumors following administration ofradioiodinated phospholipid ethers revealed only the intact agent,whereas analysis of urine and feces revealed only metabolites (Plotzke,K P, et al., J Nucl Biol Med, 1993; 37:264-272). Therefore, the reasontumors retain PLE is due to the differential clearance rates of PLE fromnormal cells versus tumor.

Extensive structure activity relationship studies resulted in thesynthesis, radiolabeling, and evaluation of over 20 phospholipid etheranalogs as potential tumor-selective imaging agents. The iodinated APCanalogues were readily labeled with all iodine radioisotopes using anisotope exchange method. These PLE analogs are specifically designed toincorporate aromatic radioiodine in order to render the molecule stabletowards in vivo deiodination. The low level of thyroid activity in allprior preclinical imaging and tissue distribution studies (on both a %injected dose/g and % injected dose/organ basis) has confirmed the invivo stability of the radioiodinated PLE analogs.

From the library of phospholipid ether (PLE) compounds, NM-324[12-(3-iodophenyl)-dodecylphosphocholine], initially showed the mostpromise in animal tumor localization studies. A variety of tumorsincluding mammary, prostate, squamous cell carcinoma, ovarian,colorectal, and melanoma were successfully visualized by scintigraphywith NM324. During initial human pharmacokinetic studies with theprototype agent, NM324, an unacceptable accumulation in liver tissue wasobserved and additional experiments to identify PLE compounds withsuperior tumor localization and background clearance properties wereperformed. Based upon this work, NM404[18-(4-iodophenyl)-octadecylphosphocholine] emerged due to its enhancedability to localize in tumor, its increased metabolic clearance from theliver, and its longer plasma half-life. A key observation documented theability of NM404 to localize in lymph node metastases, which wereclearly delineated by scintigraphy in a metastatic prostate tumor modelwithout retention in uninvolved lymph nodes.

The lead compound NM404 has now been evaluated in over 25 animal tumormodels and in every tumor model and tumor type studied so far, NM404 hasshown tumor-selective retention. Prolonged tumor retention of ¹²⁵I-NM404has been demonstrated in mice for periods of 20-60 days post-injection.Such very extensive and protracted tumor retention characteristics maysignificantly enhance the radiotherapeutic efficacy of the agent,especially for isotopes with a slow radioactive decay like e.g.iodine-125.

Extensive biodistribution data for the prototype agent ¹²⁵I-NM324 inseveral tumor models revealed tumor-to-blood ratios exceeding 8:1 atlater post-injection times. In one such example in a rat mammary tumormodel, tumor-to-normal tissue ratios reached a maximum at 96 hours witha tumor-to-blood ratio of 8.6 and tumor-to-muscle ratio of 20:1.Moreover, the heterogeneity of biodistribution of PLE-associatedradioactivity within tumor was demonstrated by microautoradiographystudies showing that the PLE radioactivity resides exclusively in viabletumor cells located toward the outer regions rather than the centralnecrotic regions. Comparative biodistribution data for NM-324 and NM-404have been obtained in SCID mouse prostate and A549 lung cancer tumormodels. These studies revealed high tumor-to-normal tissue ratios andtumor uptake exceeding 25% of the injected dose of NM-404 within thetumor, thus supporting the desire to study the biodistribution of NM404in humans.

Mechanism of Action

Metabolic Studies

Formal metabolism studies were conducted on several PLE analogsincluding NM324, the predecessor of NM404. In these studies, each agentwas examined to determine their ability to serve as substrates forenzymes associated with PLE metabolism. Three major enzymatic pathwaysare involved in the metabolism of PLE. O-Alkyl glycerol monooxygenase(AGMO) is responsible for cleavage of the alkyl ether linkage at C-1 toform either the long chain fatty alcohol or subsequently, thecorresponding fatty acid. Phospholipases C (PLC) and D (PLD), on theother hand, give rise to the glycerol or phosphatidic acid products,respectively. Using a microsomal AGMO enzyme preparation, NM324 was nota substrate for this enzyme when compared to [3H]-lyso-PAF (plateletactivating factor), which was extensively metabolized. In a similarfashion, NM324 was analyzed as a substrate for PLC isolated fromBacillus cereus and was not hydrolyzed relative to1-palmitoyl-2-[3H]-palmitoyl-L-3-phosphatidylcholine (DPPC), whichunderwent significant hydrolysis.

Finally, several PLE analogs were subjected to a phospholipase D (PLD)assay. The PLD, which was isolated from cabbage, is similar to mammalianPLD in that the cabbage form affords phosphatidylethanol-type productsin addition to phosphatidic acid when the enzymatic reaction isperformed in the presence of ethanol. Several of the PLE analogssubjected to these assay conditions did give rise to thephosphatidylethanol adduct, indicating possible interaction with PLD.The inventors believes that NM404 is a metabolic substrate to humanPhospholipase D, and that the relative absence of Phospholipase D incancer cell membranes is the underlying mechanism for tumor-selectiveretention of NM404. Although known from the literature (reference???),it is still unclear why cancers lack PLD in their membranes.

Several NM404 precursors were also subjected to in vitro metabolismstudies in various cell lines including Walker tumor cells, rat muscle(H9c2), and rat hepatocytes. In these studies, the extent of metabolismwas determined on the basis of radiolabeled products formed afterincubation for various time periods and the results normalized to cellnumber or the amount of cellular protein. Subsequent lipid extraction ofthe incubation medium and cell suspension demonstrated little generationof PLE metabolites in the Walker tumor cells whereas a significantproduction of metabolites was seen in both the muscle cells andhepatocytes over the 48 h time period studied. These results correlatenicely with in vivo biodistribution studies completed on all analogs.Although several studies have been completed, the role of metabolictrapping in the uptake and retention of radiolabeled PLE analogs intumor cells is not well defined and currently remains an active area ofexamination. The inventors believes that NM404 can enter the cellmembranes of all cells, but gets eliminated from non-cancerous cellsthrough rapid metabolism, whereas in cancer cells it gets trapped due tolack of appropriate metabolic enzymes.

PLD Assays

Due to the apparent universality of tumor retention of NM404 in animaltumor models and initial corroborative results in a human lung cancertrial, the inventors have begun to investigate the mechanism of actionof this agent. Although membrane metabolism of PLE analogs is regulatedby a variety of phospholipases, the inventors have focused initialefforts on phospholipase D (PLD) activity, based on the hypothesis thatcellular uptake and retention of NM404 is inversely related to theamount of PLD present in the tumor cell membrane relative to normalcells.

Because of these findings, preliminary evaluation of PLD proteinactivity and PLD mRNA quantification by RT-PCR assay were performed inseveral murine tumor cell lines, including the murine tumor cell linehepa-1 (hepatoma), CT26 (colorectal adenocarcinoma), and TS/A (breastadenocarcinoma) and compared to normal liver. These experiments revealedthat both PLD protein activity and mRNA levels were significantly lowerin tumor than in normal liver tissue (p<0.05, T-test) (Table 1)

TABLE 1 PLD protein activity and PLD mRNA quantification for threecancer cell lines and normal liver tissue PLD protein activity PLD mRNA(mU/fluorescence/ (μg × 10⁻⁵/ Cell/tissue μg protein/ml) 0.01 μg oftotal cDNA) Hepa-1 3.3 6.2 CT26 7.8 2.4 TS/A 2.8 4.0 Normal liver 14.112.2

As conclusion, the mechanism of selective retention of NM404 may be dueto a decrease in the membrane levels of PLD, thus precluding metabolismand clearance of NM404 from the cell. Recall that earlier enzymesubstrate assays conducted with PLD derived from cabbage indicated thatNM404 was indeed a good substrate for this enzyme. This supports thefinding of the in vitro cell culture uptake and retention study whereinit was shown that PLE analogs were sequestered by and subsequentlymetabolized by normal cells (which contained normal levels of PLD). Ifmalignant tumor cells would possess a normal complement of PLD, that theagent would have been metabolized and eliminated from the tumor cells aswell. Conversely it could be deduced that the lack of metabolism andclearance of the agent from malignant cells would support the hypothesisthat these neoplastic cells lack PLD relative to surrounding normal hostcells.

Other Studies

Mechanistic Studies with PLE Analogs: NM324 and NM404 are similar instructure to miltefosine (hexadecylphosphocholine), an antitumor etherlipid studied most extensively in Europe. The antitumor properties ofmiltefosine and several other antitumor phospholipid ether analogs havebeen demonstrated in a wide range of tumor cell lines includingprostate-, bladder-, and terato-carcinomas, murine and human leukemias,as well as lung, colon, ovarian, brain and breast cancers (Lohmeyer M,Bittman R. Drugs of the Future 1994; 19: 1021-1037). In contrast to manyanticancer drugs, these phospholipid ether analogs do not bind directlyto DNA and are not mutagenic. Although the precise antiproliferativemechanism of action has not been determined, they apparently act atseveral tumor cell sites. These compounds have been associated with avariety of cellular effects including transport, promotion of cytokineformation, apoptosis induction, and interference with a variety of keylipid metabolism and cell signaling enzymes most of which are located inthe cellular membrane. Although uncertainty remains regarding the modeof PLE uptake into cells, most evidence now supports the idea that theseether lipids are directly absorbed into cell membranes where theyaccumulate. A widespread belief is that these agents act by perturbingmembrane phospholipid metabolism; however, cellular distribution studieswith these agents have been limited by spontaneous cellularcompartmental redistribution during homogenization and subcellularfractionation procedures. In contrast to the tracer imaging doses(several μg) employed in the imaging and biodistribution studies citedby The inventors, antitumor effects are seen only at doses generallyexceeding 150 mg per day (Planting A S, Stoter G, Verweij J. EuropeanJournal of Cancer, 1993; 29A:518-9; Verweij J, Planting A, van der BurgM, Stoter G. Journal of Cancer Research & Clinical Oncology, 1992;118:606-8; Muschiol C, et al. Lipids 1987; 22:930-934).

Mechanism of Action

The prevailing mechanism for action is that phospholipid ethers such asNM404 become trapped in malignant tumor cell membranes because of theirinability to become metabolized and eliminated. Extraction of tumorsfollowing administration of radioiodinated phospholipid ethers showedthe presence of only the intact agent, whereas analysis of the urine andfeces revealed only metabolites. Thus, it is the differential clearancerates of phospholipid ethers from normal cells versus tumor cells thatform the basis of this concept. Preliminary results obtained in over 27xenograft and spontaneous tumor models have universally shown NM404 toundergo selective and prolonged retention in tumors.

Isotope Selection for Therapy

The inventors believe that iodine-125 is the most appropriateradioisotope for combination with the NM404 targeting backbone, since:

The long isotope half-life of iodine-125 matches perfectly with the longand stable tumor retention of NM404, delivering therapeutic radiationdoses for an extended period of time.

The effect of iodine-125 is caused by both, low-energy gamma/X-rayirradiation and by Auger electrons, all of which are of very limitedtreatment distance. Since NM404 is taken up into the tumor, iodine-125can effectively deliver a tumor dose but it is sparing surroundinghealthy tissue.

Iodine-125 has Auger electrons as one of its radio-decay by-products(FIG. 2). Auger electrons cause a pronounced biological effect, but havea very short treatment distance. Since NM404 is taken up directly intoall cell membranes of cancer cells (including the nuclear membrane), thetreatment distance to the DNA is very low. It may effectively allowAuger electrons to be a major contributor to I-125-NM404 treatmenteffects.

The iodine-125 isotope is used for formulation in this applicationbecause it exhibits favorable characteristics for cancer radiotherapy,with properties listed below:

Gamma Irradiation (1) Radioisotopic half-life: 59.43 days (2) Gammaenergy: 35.5 keV (3) X-Ray energy: 27.5-31.7 keV (4) Radiationhalf-distance: 0.02 mm lead; 2 cm in tissue

Auger Electrons (1) Energy of radiation: 1 keV (2) Number of Augerelectrons: Up to 21 per gamma decay (3) Radiation half-distance: 1-10 nm

It should be noted that although iodine-125 has great utility forin-vivo biodistribution studies and dosimetry extrapolation fordiagnostic imaging, it is poorly suited for either whole-body planarimaging or in vivo scintigraphic quantification of tissue concentrationsof NM404. However, iodine-124 offers superior characteristics forquantitative determination of in vivo tissue concentrations. Thus,124-I-NM404 will find utility for in vivo pharmacokinetics andbiodistribution studies but has no radiotherapeutic effect. Iodine-131emits both beta and gamma radiation that produces a therapeutic effect.Although I-131-NM404 could potentially be used for radiotherapy, theinventors believe I-125 to be the more optimal isotope because its lowerenergy radiation has a shorter radiation half distance than I-131 andthus hypothesize that it will produce less damage to healthy tissue.Therefore, it was decided that I-125-NM404 would be used for allclinical radiotherapy studies performed in order to reduce the potentialfor collateral damage to healthy tissues.

Due to its 60-day physical half-life and low energy 35 keV photonemission,—iodine-125 is suitable for imaging experiments in mice andrats. Iodine-125 also provides therapeutic efficacy when used inpermanent prostate brachytherapy implants (“brachytherapy seeds”). Themajor advantage of 125I is that all the photons are of low energy,insuring very limited exposure of normal tissues surrounding the tumor.The major difference between brachytherapy seeds containing iodine-125and I-125-NM404 are the effect of Auger electrons. Since brachytherapyseeds have a metal capsule around the iodine-125, only the low-energygamma and X-ray are of therapeutic value, and Auger electrons areeliminated by the metal capsule. In difference, I-125-NM404 is taken upinto the cancer cell membranes (including into the nuclear membrane), sothat Auger electrons can have a major contribution to the therapeuticeffect.

Although iodine-131 has been used with great efficacy in the treatmentof thyroid cancer, a significant disadvantage of iodine-131 is thatthere is a higher energy gamma emission which could actually exposeadjacent surrounding tissues to more radiation than would occur withiodine-125.

Dosimetry

Dosimetry estimation regarding I-125-NM404 for an adult female patientswere calculated based on SPECT imaging data using female non-tumorbearing rats following administration of I-131-NM404. The results arelisted below:

MIRD extrapolation of 125I-NM404 rat tissue distribution data initiallyafforded a 5 Rad limiting dose to the adrenals and bladder wall of 2 mCiof ¹³¹I-NM404. Accordingly the inventors have conducted the preliminarypharmacokinetic studies in human lung cancer patients at 1 mCi. Similarextrapolative calculation of the dosimetry for ¹²⁴I-NM404 afforded asimilar 2 mCi dose level again based on projected dosimetry to theadrenals and bladder wall. Preliminary human data (5 patients), however,indicate very little uptake and retention of the agent into anyabdominal organ (including the bladder and adrenals) with the exceptionof the liver which returns to background levels within 11 days. Althoughthese results are not yet complete, it is likely that the actualacceptable ¹²⁴I-NM404 dose will exceed 4 mCi.

MIRDOSE (IBM PC VERSION 3.1 - AUGUST 1995) Radiation Dose Estimates forthe ADULT FEMALE for 125-I-NM404 Assumptions: Predicted Residence Timefrom I-131-NM404 female rat biodistribution data TOTAL DOSE PRIMARYSECONDARY TARGET ORGAN mGy/MBq rad/mCi CONTRIBUTOR % CONTRIBUTOR %  1)Adrenals 7.34E−02 2.72E−01 Adrenals 100.0% 0.0%  2) Brain 5.96E−032.20E−02 Rem. Body 100.0% 0.0%  3) Breasts 5.96E−03 2.20E−02 Rem. Body100.0% 0.0%  4) Gallbladder Wall 5.96E−03 2.20E−02 Rem. Body 100.0% 0.0% 5) LLI Wall 5.96E−03 2.20E−02 Rem. Body 100.0% 0.0%  6) Small Intestine7.42E−02 2.75E−01 Small Int. 96.0% Rem. Body 4.0%  7) Stomach 5.96E−032.20E−02 Rem. Body 100.0% 0.0%  8) ULI Wall 5.96E−03 2.20E−02 Rem. Body100.0% 0.0%  9) Heart Wall 3.12E−02 1.15E−01 Heart Wall 100.0% 0.0% 10)Kidneys 5.09E−02 1.88E−01 Kidneys 100.0% 0.0% 11) Liver 4.66E−021.72E−01 Liver 100.0% 0.0% 12) Lungs 6.43E−02 2.38E−01 Lungs 100.0% 0.0%13) Muscle 2.54E−02 9.40E−02 Muscle 100.0% Uterus 0.0% 14) Ovaries4.10E−02 1.52E−01 Ovaries 100.0% 0.0% 15) Pancreas 5.96E−03 2.20E−02Rem. Body 100.0% 0.0% 16) Red Marrow 2.75E−02 1.02E−01 Red Marrow 95.2%Rem. Body 4.8% 17) Bone Surfaces 1.84E−02 6.80E−02 Red Marrow 82.1% Rem.Body 17.9% 18) Skin 5.96E−03 2.20E−02 Rem. Body 100.0% 0.0% 19) Spleen4.79E−02 1.77E−01 Spleen 100.0% 0.0% 21) Thymus 5.96E−03 2.20E−02 Rem.Body 100.0% 0.0% 22) Thyroid 2.98E−01 1.10E+00 Thyroid 100.0% 0.0% 23)Urin Bladder Wall 1.13E−02 4.18E−02 Urinary Bl 73.7% Rem. Body 26.3% 24)Uterus 4.78E−02 1.77E−01 Uterus 100.0% 0.0% 27) Total Body 1.55E−025.75E−02 Muscle 48.8% Rem. Body 23.7% 28) EFF DOSE EQUIV 4.93E−021.82E−01 Remainder 35.8% Gonads 20.8% 29) EFF DOSE 4.32E−02 1.60E−01Thyroid 34.5% Gonads 19.0% Units of EDE and ED are mSv/MBq or rem/mCi.RESIDENCE TIMES: Adrenals 8.99E−02 Ovaries 3.95E−02 hr Small Intestine4.68E+00 Red Marrow 3.83E+00 hr Heart Wall 6.54E−01 Spleen 6.29E−01 hrKidneys 1.22E+00 Thyroid 4.44E−01 hr Liver 5.71E+00 Urinary Bl Cont2.33E−01 hr Lungs 4.50E+00 Uterus 3.34E−01 hr Muscle 3.78E+01 Remainder1.83E+01 hr For 5 rads to Adrenals:  18 mCi For 3 rads to Ovaries:  20mCi For 5 rads to Thyroids: 4.5 mCi

As a result of these dosimetry estimation, the following point have tobe considered:

These results were based on a worst-case scenario of no excretion ofNM404 from the body. The dosimetry data were calculated from SPECTimaging in rats using I-131-NM404 and then converted to I-125-NM404. Theadrenal gland seems to be the dose-limiting or critical organ forradiation exposure. In the SPECT imaging experiments the thyroids ofrats were unblocked. Thus, the thyroid calculations should be evaluatedwith caution.

Pharmacology Summary

Comprehensive summary of the synthesis and the biological properties ofNM404 is discussed in U.S. Patent Application No. 60/593,190 filed onDec. 20, 2004 U.S. application Ser. No. 10/906,687 filed on Mar. 2, 2005and U.S. Provisional Application No. 60/521,166, filed on Mar. 2, 2004,all of which are incorporated herein by reference for all purposes.

The inventors' approach is to design small-molecule carrier moleculeswhich are capable of selectively delivering a diagnostic or therapeuticprobe to the desired target tissue capitalizes on unique biochemical orpharmacological properties of molecules displaying a high degree oftissue or tumor selectivity.

It was initially observed that a variety of animal and human tumorscontained much higher concentrations of naturally occurring ether lipidsin the cell membranes than normal tissue. It was hypothesized thatphospholipid ether analogs could accumulate in tumor cells due to theirlower capacity to metabolize these lipids. The inventors have pursuedradioiodinated phospholipid ether (PLE) analogs as potentialtumor-selective imaging agents. Several PLE analogs have exhibited astriking universal ability to selectively localize in a wide variety oftransplanted rat, mouse, and human tumor models.

The prevailing hypothesis is that phospholipid ethers become trapped intumor membranes because of their inability to be metabolized andcleared. Indeed, tumor analysis following administration ofradioiodinated phospholipid ethers showed the presence of only theintact agent, whereas analysis of the normal tissues (liver and muscle),urine, and feces revealed only metabolites. Thus, it is the differentialclearance rates of phospholipid ether analogs from normal cells versustumor cells that form the basis of this targeting concept.

Preclinical Studies with First Generation PLE Analogs:

Phospholipid ethers can easily be labeled with iodine radioisotopesusing radiolabeling methods developed in the labs. The iodophenylphospholipid ether analogs are specifically designed so that theradioiodine affixed to each molecule is stable to facile in vivodeiodination. It was found that any chemical modification of thephosphocholine moiety or shortening the chain length of theiodophenylalkyl moiety to less than 8 methylenes resulted in little orno tumor uptake. The inventors have now synthesized over 20 radiolabeledPLE compounds and tested in vitro and in vivo. Two of these, namelyNM-294 and NM-324 [12-(3-iodophenyl)-dodecyl-phosphocholine], initiallyshowed the most promise in animal tumor localization studies. Theseprototype compounds, labeled with iodine-125, selectively localized intumors over time in the following animal tumor models; 1) Sprague-Dawleyrat-bearing Walker 256 carcinosarcoma; 2) Lewis rat-bearing mammarytumor; 3) Copenhagen rat-bearing Dunning R3327 prostate tumors; 4)Rabbits-bearing Vx2 tumors; and 5) athymic mice-bearing human breast(HT39), small cell lung (NCI-69), colorectal (LS174T), ovarian(HTB77IP3), and melanoma tumors. Optimal tumor localization of theseagents takes from one to several days due to the more rapid clearance ofthe radioactivity from normal tissues relative to tumor.

Certain PLE compounds discussed in the following paragraphs are shown inFIG. 1.

Clinical Evaluation of NM324:

Although first generation compounds NM-324 and NM-294 displayed similaranimal tumor localization characteristics, NM-324 was easier tochemically synthesize and was thus selected as the lead compound forinitial clinical studies. Although images obtained in several human lungcancer patients detected tumors, images were complicated by high liverradioactivity (FIG. 3).

Second Generation PLE Analogs:

In order to decrease liver uptake and prolong the plasma phase, theinventors examined 9 structural analogs of NM-324 to identify agentsthat would display improved tumor-to-background tissue ratios withdecreased liver uptake. The new PLE analogs were synthesized andradiolabeled with ¹²⁵I for initial image analysis in Copenhagen ratsbearing Dunning R3327 prostate tumors. Based upon this initial screen,NM-404 not only exhibited much lower liver activity than its predecessorNM324 but also maintained prolonged tumor retention (FIG. 4. NM404 wastherefore selected to undergo further imaging and biodistributionanalysis in a variety of animal-tumor models.

Accordingly, NM404 has now been evaluated in over 20 xenograft andspontaneous animal tumor models as shown in Table 2. In all tumor modelsthe agent displayed significant tumor uptake and retention regardless oflocation. Although tumor retention can be explained by a lack ofmetabolic phospholipase enzymes in the tumor cell membranes, the exactmechanism of tumor cell uptake is unknown. Furthermore, the inventorsknow the agent does not localize in benign intestinal adenomas (polyps)so it was desirable to further evaluate the propensity of the agent tolocalize in an intermediate stage of tumorigenesis, namely hyperplasia.The inventors are currently evaluating the selectivity of NM404 in aunique mouse tumor model developed at Wisconsin, wherein bothpreneoplastic hyperplasias and malignant adenocarcinomas formspontaneously in the mammary gland. Preliminary results in this modelindicate that the agent is not taken up and retained in preneoplasticlesions, and thus, appears to be exclusively retained by malignant tumorcells. If this initial observation is validated, then via genomic and orproteomic analysis the inventors may be able to identify a key geneticdifference between malignant tumors and their non malignant predecessorcells. This could identify a completely new molecular therapeutic targetthat may be universal for all types of cancer.

The table below summarizes the wide variety of cancer types and tumorlines that have been evaluated regarding accumulation by NM404.

TABLE 2 Tumor models examined with NM404 NM404 Tumor Tumor Model SpeciesType Localization* Human Tumor Xenografts Prostate PC-3 SCID MouseAdenocarcinoma Yes Lung A-549 (NSCC) SCID Mouse Adenocarcinoma Yes LungNCI H-69 (Oat Cell) Nude Mouse Adenocarcinoma Yes Adrenal H-295 SCIDMouse Adenocarcinoma Yes Adrenal RL-251 SCID Mouse Adenocarcinoma YesMelanoma A-375 Nude Mouse Adenocarcinoma Yes Colon LS-180 Nude MouseAdenocarcinoma Yes Ovarian HTB-77 Nude Mouse Adenocarcinoma Yes AnimalTumor Xenografts Mammary MCF-7 Rat Adenocarcinoma Yes Prostate MatLyLuRat Adenocarcinoma Yes Walker-256 Rat Carcinosarcoma Yes Recent RodentTumor Models TRAMP prostate Transgenic mouse Adenocarcinoma Yes LuCaPprostate Mouse xenograft Adenocarcinoma Yes Liver CT-26 Mouse xenograftColorectal Yes adenocarcinoma TGFα Hepatoma Transgenic mouse HepatomaYes Min Mouse Intestinal Transgenic mouse Adenocarcinoma Yes MelanomaB16 Mouse xenograft Adenocarcinoma Yes SCC1 and 6 Nude mouse Squamouscell carcinoma Yes xenograft Mammary Adenocarcinoma Apc/^(min+) mouseAdenocarcinoma Yes Mammary SCC Apc/^(min+) mouse Squamous cell carcinomaYes Glioma L9 and CNS-1 Rat xenograft Glioma Yes Pancreas c-myc/k-rasTransgenic mouse Ductal/acinar Yes Retinoblastoma Transgenic mouseBlastoma Yes Cervical Transgenic mouse Adenocarcinoma Yes Mammaryalveolar hyperplasia Apc/^(min+) mouse Hyperplasia No Intestinal adenomaApc/^(min +) mouse Hyperplasia No *Localization defined as >5% injecteddose per gram tumor basis based on tissue distribution data. No decaycorrected tumor clearance was observed from 14 to 80 days in thesemodels using ¹²⁵I-NM404.

Tissue Distribution and Kinetics

Consistently, NM404 has been found to be retained in tumor tissue forlong and extended periods of time. Tumor concentrations are almoststable for many weeks following administration of NM404, showing slowelimination from cancerous tissue over time. In contrast, NM404 iseliminated from normal tissue within a few days reaching very lowlevels.

Additionally, NM404 was designed to have a long blood half life. Thisensures prolonged exposure of NM404 to the tumor tissue and ensuresuptake of up to 10-25% of the injected dose into the tumor tissue. Theinventors believe that it is very important for a radiotherapy compoundto have a large portion of the injected dose accumulating in the tissueof interest. As a consequence of the long blood half life, NM404 willcontinuously accumulate in tumor tissue over time. An example of thispattern is provided in FIG. 5. This may lead to a delayed onset of thetherapeutic effect until the accumulation of NM404 into the tumor tissuehas been continuing for several days or weeks.

Blood Plasma Kinetics

The first-generation prototype compound NM324 was found to have aelimination half life time in plasma of 2.43 hours in rats. Bycomparison, the lead compound NM404, has a elimination half life time ofroughly 209 hours in rats (distribution phase half life is 4.86 hours).

Radiotherapeutic Study of I-125-NM404

During the course of mouse tumor uptake and retention studies with“imaging” doses (15-20 μCi/20 g mouse) of ¹²⁵I-labeled NM404, severalapparent therapeutic responses have been observed (unpublished results).In an Apc^(Min)/+ mouse mammary tumor model it has generally been notedthat tumor growth remains static following a single intravenousinjection of NM404. Some of these animals also lost all hair abovelarger mammary tumors at around 8 days after injection. Moreover, thesemice also get intestinal tumors and usually suffer from intestinalbleeding resulting in severe anemia, which renders their feet white. Dr.Moser noted that the feet of these mice had reverted to a pink coloraround 5 days after a single injection of NM404. Upon eventualdissection of these animals, it was noted that only a very few, if any,of the expected 20 or so intestinal tumors usually found at this ageactually remained. The “white to pink feet” phenomenon was also observedin a separate, but more aggressive, mouse intestinal adenocarcinomamodel, wherein dissection at 12 days following NM404 administration,again revealed that most, if not all, of the expected intestinal tumorswere gone. After 21 days In both intestinal models, animals thatreceived NM404 easily outlived their untreated litter mates. Anothercompelling example of tumor regression is illustrated in FIG. 6. Twolitter mates each received SCC1 and SCC6 xenografts in their left andright flanks, respectively. One mouse received a single injection of¹²⁵I-NM404 (20 μCi). The mouse that didn't receive NM404 died 21 dayslater, whereas the tumors in the treated mouse regressed significantlyand the animal was quite healthy 80 days after injection. Thesecoincidental findings were reconfirmed in two separate age-matchedgroups each involving more than 6 mice. Although these observations with¹²⁵I-NM404 are anecdotal at this point, they do seem to stronglyindicate potential for radiotherapy applications particularly if labeledwith iodine-131. Ongoing quantitative tumor uptake and retention studiesin several animal tumor models will also provide sufficient data toinitiate a comprehensive dosimetry analysis for this agent in order toestimate its true radiotherapeutic potential.

Due to its 60-day physical half-life and low energy 35 KeV photonemission, iodine-125 is suitable for imaging experiments in mice andrats. Iodine-125 also affords therapeutic characteristics. In oneimaging experiment (FIG. 6), 2 nude mice were each inoculated withsubcutaneous squamous cell lines SCC1 and SCC6 tumor cell implants onopposing flanks. SCC1 and SSC6 cells were used because one isradiosensitive relative to the other. After 14 days when the averagetumor size was approaching 0.5 cm in diameter, one of the mice received20 μCi of I-125-NM-404 and the other one receive unlabeled NM404 in anequal mass dose. The mouse receiving the unlabeled cold compound had tobe euthanized 20 days after injection due to both tumors reaching thetermination size limit as defined in the animal use protocol. Bothtumors in the ¹²⁵I-NM404 mouse regressed dramatically and unexpectedlyover the course of several weeks (FIG. 6) after a single injection of animaging dose of NM404. This mouse never did reach terminal tumor sizeand the mouse was actually euthanized after 90 days in order to collecthistology sections.

Preliminary Report of Ongoing Tumor Therapy Studies

Preclinical study-preliminary results:

Efficacy of Single Injection of I-125 Labeled SF404A in an A549 SCIDMouse Model

Purpose and Rationale:

To determine an effective dose for radiotherapy using single injectionof I-125 labeled SF404A in a xenograft tumor model

Tumor Model:

A549 (human non-small cell lung cancer, NSCLC, from ATCC) are maintainedin Ham's F-12K media supplemented with 10% fetal bovine serum. Tumorcell suspension (1×10⁶ cells in phosphate-buffered saline) is injecteds.c. into the right flank of female SCID mice (6-8 weeks,C.B-17/IcrHsd-Prkcd^(scid), Harlan). Animals are given free access tofood and water, tumor growth and animal weight are monitored. Uponreaching 4-5 mm in diameter, mice will be divided into groups of 6 fortherapy study.

Dosing:

Single injection of SF404A at Day 0

Groups:

-   -   (1) 5 study groups: 4 treatment+1 control    -   (2) N=6 per group    -   (3) 4 acute dose levels: 50, 150, 250 and 500 μCi per mouse    -   (4) Control group dosed with equivalent mass amount of NM404:    -   (5) Control: 0 μCi per mouse (“cold” NM404)

Efficacy Assessments:

-   -   (1) Tumor size (caliper) measured once a week.    -   (2) Survival    -   (3) General appearance (activity, alertness, mobility)    -   (4) Until 10 weeks post-injection or until no animal in the        control group is alive, whatever comes first    -   (5) Histopathology assessment of tumor or residual tumor site.

Photograph of tumor at Day 0, 30, and 60 for each animal.

The purpose of the study was to determine an effective dose forradiotherapy using single injection of I-125-NM404 (formulation SF404A)in a xenograft tumor model.

The tumor model used for this study was A549, a human non-small celllung cancer, (NSCLC) obtained from ATCC (Manassas, Va.). The tumor cellsare maintained in Ham's F-12K media supplemented with 10% fetal bovineserum. Tumor cell suspension (1×10⁶ cells in phosphate-buffered saline)is injected s.c. into the right flank of female SCID mice (6-8 weeks,C.B-17/IcrHsd-Prkcd^(scid), Harlan). Animals are given free access tofood and water. Upon reaching 5-10 mm in tumor diameter, mice will beenrolled in the study.

A single intravenous injection of SF404A was performed at Day 0. For thecontrol group, cold compound (C-NM404) has been injected. For treatmentgroups I-125-NM404 has been injected.

The study contained 5 groups; 4 treatment groups (n=6/group) and 1control group (n=9). The iodine doses for the treatment groups were 50,150, 250 and 500 μCi per mouse. The control group was dosed with anequivalent mass amount of C-NM404 (0 μCi).

For assessment of efficacy of treatments, the following assessments wereperformed:

-   -   (1) Tumor size (caliper) measured once a week    -   (2) Survival    -   (3) General appearance (activity, alertness, mobility)    -   (4) Digital pictures of tumor-bearing mouse

Assessments were made until 10 weeks post-injection or until no animalin the control group is alive, whatever comes first. Histopathologyassessment of tumor or residual tumor site was performed at the end ofthe study.

-   -   (1) 50 μCi group: 6 animals were enrolled;    -   (2) 150 μCi group: 5 animals were enrolled;    -   (3) 250 μCi group: 6 animals were enrolled;    -   (4) 500 μCi group: 6 animals were enrolled.

As part of this preliminary report, the inventors include the followinganimals in the analysis:

(1) Control: n = 7 (2)  50 μCi group: n = 4 (3) 150 μCi group: n = 5 (4)250 μCi group: n = 6 (5) 500 μCi group: n = 5

The baseline tumor volumes (in mm³) for each group were:

Average tumor volume SD Control 1152 1314 50 μCi group: 198 138 150 μCigroup: 1699 1251 250 μCi group: 360 156 500 μCi group: 1041 673

Since the tumor volumes of the control group and the 150 μCi group wassubstantially larger than the other groups and from what was previouslyplanned, a complete re-enrollment of both groups with smaller tumorsizes has been initiated. As far as the tumor volumes for the 500 μCigroup, the inventors believe that the study may be biased against thisgroup. Because of necrosis and inefficient blood supply, larger tumorsmay not respond as well to I-125-NM404 treatment.

The following average tumor volume for each group was recorded over the10-week assessment period:

Control 50 μCi 150 μCi 250 μCi 500 μCi Week (n = 7) (n = 4) (n = 5) (n =6) (n = 5) 0 1152 198 1699 360 1041 1 1289 599 1847 964 2 1667 691 2430756 1281 3 2418 987 2758 821 1110 4 3169 2283 4144 1300 5 3763 3977 44781626 6 4971 4951 4668 1735 7 6207 6999 4817 1697 8 6253 8188 6049 9 71759978 7780 10 7068 8342 6894

This is also reflected in the FIG. 10

In summary, control animals show rapidly growing tumors over the 10-weekassessment period. This confirms that the compound itself C-NM404 has nosubstantial effect on tumors growth. The 50 μCi dose group did not showany difference to control animals, hence this seems to be an ineffectivedose in this animal model. The 150 μCi dose group shows no treatmenteffect as well, however (as was pointed out before) this dose groupstarted with unusually large tumors at Day 0, however this may showdifferent results with this dose group of 150 μCi with smaller tumors.

Preliminary data indicates that both the 250 and 500 μCi show asubstantial and prolonged treatment effect. Tumor volumes are stable andsame tumors appear “collapsed” (the tumor surface has caved in, as shownin FIG. 7). Additionally, hair above the tumors fells off confirmingsubstantial accumulation of radioactivity in these tumors.

Other Ongoing Preclinical Tumor Therapy Studies

Efficacy of fractionated dose vs single injection of I-125 labeledSF404A in an A549 SCID mouse model

This preclinical study assesses fractionating the 150 μCi dose byinjecting 3 weekly 50 μCi doses instead of one single 150 μCi dose asgiven in the previous study.

Purpose and Rationale:

To determine the radiotherapeutic efficacy of a fractionated dose ofI-125 labeled SF404A vs a single injection of an equivalent total dosein a xenograft tumor model This study is an adjunct to CLTR-Pre-05-001,which contains the 150 μCi single dose used for comparison.

Tumor Model:

A549 (human non-small cell lung cancer, NSCLC, from ATCC) are maintainedin Ham's F-12K media supplemented with 10% fetal bovine serum. Tumorcell suspension (1×10⁶ cells in phosphate-buffered saline) is injecteds.c. into the right flank of female SCID mice (6-8 weeks,C.B-17/IcrHsd-Prkcd^(scid), Harlan). Animals are given free access tofood and water, tumor growth and animal weight are monitored. Uponreaching 4-5 mm in diameter, mice will be divided into groups of 6 fortherapy study.

Dosing:

Single injection of I-125-SF404A at Day 0 vs a fractionated dose ofI-125-S404A

Groups:

2 study groups: 1 Fractionated dose, 1 Single injection

-   -   (1) N=6 per group    -   (2) 150 μCi (3×50 μCi) per mouse by fractionated injection, at 1        week intervals from day 0    -   (3) 150 μCi per mouse by single injection

Efficacy Assessments:

-   -   (1) Tumor size (caliper) measured once a week.    -   (2) Survival until 10 weeks post-injection or until no animal in        either group is alive, whatever comes first    -   (3) General appearance (activity, alertness, mobility)    -   (4) Histopathology assessment of tumor or residual tumor site.    -   (5) Photograph of tumor at Day 0, 30, and 60 for each animal.

Efficacy of Single Injection of I-125 Labeled SF404A in a PC-3 (ProstateCancer) SCID Mouse Model

Purpose and Rationale:

To determine an effective dose for radiotherapy using single injectionof I-125 labeled SF404A in a prostate cancer xenograft tumor model

Tumor Model:

PC-3 (human prostate cancer, from ATCC) are maintained in Ham's F-12Kmedia supplemented with 10% fetal bovine serum. Tumor cell suspension(1×10⁶ cells in phosphate-buffered saline) is injected s.c. into theright flank of male SCID mice (6-8 weeks, C.B-17/IcrHsd-Prkcd^(scid),Harlan). Animals are given free access to food and water, tumor growthand animal weight are monitored. Upon reaching 4-5 mm in diameter, micewill be divided into groups of 6 for therapy study.

Dosing:

Single injection of SF404A at Day 0

Groups:

-   -   (1) 5 study groups: 4 treatment+1 control    -   (2) N=6 per group    -   (3) 4 acute dose levels: 50, 150, 250 and 500 μCi per mouse    -   (4) Control group dosed with equivalent mass amount of NM404: 0        μCi per mouse (“cold” NM404)

Efficacy Assessments:

Tumor size (caliper) measured once a week.

Survival:

until 10 weeks post-injection or until no animal in the control group isalive, whatever comes first

-   -   (1) General appearance (activity, alertness, mobility)    -   (2) Histopathology assessment of tumor or residual tumor site.    -   (3) Photograph of tumor at Day 0, 30, and 60 for each animal.

This preclinical study duplicates the previous study single injectionstudy but with a different tumors model.

Clinical Evaluation for Imaging of Prostate and NSCLC Using aPhospholipid Ether Analog, NM-404.

Safety assessment in normal male subjects healthy normal female subjectswere carried out and it was determined that a dose level of 3 μg/kg ofC-NM-404 is safe to administer to male subjects. This dose is 10 foldthe 0.3 μg/kg dose of ¹³¹I-NM404 anticipated to be used for imaging inpatients with metastatic prostate cancer. A dose level of 3 μg/kg ofC-NM-404 is safe to administer to female subjects. This dose is 10 foldthe 0.3 μg/kg dose of ¹³¹I-NM404 anticipated to be used for imaging inpatients with metastatic lung cancer.

Imaging Characteristics of NM-404 in Patients with NSCLC

Metastatic Non-Small Cell Lung Cancer Subjects:

Written informed consent will be obtained. ¹³¹I-NM404 at a dose of 0.3μg/kg will be infused over 10 minutes. The aqueous solution containing¹³¹I-NM404 will be prepared using sterile technique in Dr. JameyWeichert's laboratory at the University of Wisconsin. The preparationdispensed by the Nuclear Pharmacy will be certified as sterile andpyrogen-free. The subject will be monitored for adverse reactions duringand after the infusion. Vital signs will be checked immediately afterthe infusion, and at 60 minute intervals up to four hourspost-injection. They will be observed for adverse events throughout thistime. The subjects will return 24, 48, 72, 96, 120, 144 hours and 30days after the infusion. NM404 Pet scans will be obtained at 4, 8, 24,48 and 96 hours post injection. Vital signs will be monitored at thesetimes. Any adverse reactions experienced by the subjects will berecorded. All subjects will receive SSKI oral solution beginning one dayprior to the 131I-NM404 infusion and continue for 7 days to reducethyroid exposure to free radioiodine. Serum pharmacokinetics will bedrawn pre-infusion, 5, 10, 30, 60, 120, 240, 360 minutes post-injectionand at 24, 48, 72 and 96 hours post injection. Sequential 24 hour urinecollections for 0-24, 24-48, 48-72 and 72-96 hours post injection willbe obtained.

Plasma pharmacokinetics confirmed an average 113.1 hours (SEM=7.9 h)elimination half life time for NM404.

An average of 3.4% (range from 0.9% to 9.8%) of injected dose waseliminated via the kidneys with 96 hours of intravenous administrationof NM404. A dose level of 0.3 μg/kg of 131I-NM404 was safe to administerto subjects with advanced non-small cell lung cancer. The eliminationplasma half life time of NM404 was found to be 113.1 hours and theurinary elimination was found to be roughly 3.4% within 96 hourspost-injection.

Prostate Cancer

The present invention provides preliminary data regarding the use of thesecond-generation PLE analog, NM404, in imaging patients with prostatecancer. This agent, currently under investigation at the University ofWisconsin, is selectively retained in tumors in high levels, and hashigh sensitivity and specificity in preclinical models. It has passedacute toxicology testing in both rats and rabbits at >1000 times theanticipated human imaging dose, and the unlabeled agent was administeredat 10 times the anticipated imaging mass dose to 10 normal volunteers atthe University of Michigan and University of Wisconsin to documentsafety. The inventors hypothesize that imaging with NM404 willultimately prove as sensitive as imaging with FDG, will also be morespecific, may afford therapeutically utility and due to its relativelylong half-life be available in virtually every PET facility regardlessof location.

The biodistribution, kinetics, optimal imaging times, and dosimetry of¹³¹I-NM404 are currently being evaluated in a pilot study in patientswith lung cancer (NSCLC) at UWCCC. This agent has also been evaluatedpreclinically in a metastatic prostate tumor model in which lymph nodemetastases were clearly delineated by scintigraphy following intravenousadministration of NM404 but, pertinently, the tracer was not retained byuninvolved lymph nodes. Selectivity of NM404 for malignant cells isparticularly relevant in prostate cancer as conventional tumor markerssuch as PSA may also be elevated in a variety of benign disease statesincluding prostatitis and benign prostatic hypertrophy. Furthermore,selective uptake and prolonged retention by tumor cells supports a rolefor NM404 as a tumor-selective diagnostic and therapeutic agent.

The preliminary results will provide the preliminary data for asubsequent study designed to more accurately estimate the predictivepower of NM404 for staging and/or following response to therapy inprostate cancer. In addition since NM404 has high tumor uptake, thisagent also has the potential to be developed as a therapeutic agent whencoupled with higher doses of ¹³¹I, ¹²⁵I, another halogen astatine.Iodine-125 would be especially desirable in prostate cancer patients dueto its short Auger electron path length in tissue, which wouldtheoretically minimize radiation effects in neighboring normal tissueslike the rectum. In a therapeutic sense, the 60 day half-life ofiodine-125 matches exceedingly well with the prolonged tumor retentionproperties of NM404.

Targeting Tumor Cells

One approach to the development of sensitive, more available imagingexams is to design carrier molecules which are capable of selectivelydelivering a radiopharmaceutical probe to the desired target tissue. Theapproach has been to capitalize on the unique biochemical andpharmacological properties of phospholipid ether analogues such asNM404, which displays a high degree of tumor selectivity. Phospholipidsare an essential component of cellular membranes where they impartstructural integrity and are heavily associated with a variety of cellsignaling processes. Phosphatidylcholine, commonly known as lecithin, issuch an example. Phospholipid ethers, on the other hand, represent aminor subclass of phospholipids that also reside in membranes. As thename implies, these lipids contain an ether rather than an ester linkageat the C-1 position. Platelet-activating factor (PAF) represents one ofthe better known phospholipid ethers. As described above, tumors retainPLE is due to the differential clearance rates of PLE from normal cellsversus tumor.

While initiating human pharmacokinetic studies with the prototype agent,NM324, ongoing experiments to identify PLE compounds with superior tumorlocalization and background clearance properties were performed. Basedupon this work, NM404 [12-(4-iodophenyl)-octadecylphosphocholine] wasselected due to its enhanced ability to localize in tumor, its increasedmetabolic clearance from the liver, and its longer plasma half-life. Ina key experiment documenting the ability of NM404 to localize inmetastases, lymph node metastases were clearly delineated byscintigraphy in a metastatic prostate tumor model following intravenousadministration of NM404, but the tracer was not retained by uninvolvedlymph nodes.

Comparative scintigraphic imaging results for NM324 and NM404 in PC-3prostate tumor-bearing SCID mice revealed high tumor-to-normal tissueratios and significant decreases in background abdominal and liverradioactivity with NM404 (FIG. 19). This agent has now been evaluated in27 animal tumor models and it is clear that once the agent enters tumorcells, it reaches a metabolic dead end and becomes trapped. Prolongedtumor retention of this agent is demonstrated in a human adrenal tumorxenograft implanted into SCID mice (FIG. 20). Using ¹²⁵I-labeled NM404,the inventors have been able to image mammary and prostate tumors inmice in excess of 60 days. Prolonged tumor retention characteristicssignificantly enhance the radiotherapeutic efficacy of the agent.

Recent imaging and biodistribution studies performed in rodent modelsaimed at determining the uptake characteristics in a wide variety ofxenograft and spontaneous (endogenous or transgenic) tumor types aresummarized above. These agents have displayed selective localization andprolonged retention in every malignant tumor regardless of anatomiclocation (including lymph nodes) studied to date.

Preliminary Scintigraphic Imaging Results with NM404 in ProstateTumor-Bearing Mice:

In a preliminary experiment to show that NM404 localizes in mouseprostate tumors, TRAMP mice were scanned on a Bioscan AR-2000 radioTLCscanner (modified in the lab for mouse imaging) from 1-8 days after tailvein injection of ¹²⁵I-NM404 (15 μCi). Following in vivo imaging ofanesthetized mice, the prostate tumors were removed immediately, andimaged ex vivo on the same scanner (equipped with high resolution 1 mmcollimator and 2-D acquisition and analysis software) in order to avoidtissue attenuation associated with the low energy of iodine-125 (FIG.21). Although the number of prostate tumor-bearing animals was small(n=4), preliminary images and tumor-to-background data indicated uptakeof NM404 into prostate tumor but not benign hyperplasia found in thismodel. In an attempt to simulate bone metastases another experiment wasperformed wherein human PC-3 tumor cells were implanted into the tibiaof immune compromised NUDE mice (n=6) according to a recent reports.Following tumor cell inoculation, mice were scanned serially byhigh-resolution microCT in order to monitor bone tumor development (FIG.22). Once bone deterioration was detected, iodine-125 labeled NM404 wasadministered intravenously via tail vein. Mice were scanned forradioactivity and by microCT 4 days post NM404 administration.Radioactivity scans were fused with microCT scans (FIG. 23) tocorroborate NM404 radioactivity and tumor location.

Biodistribution Data

Extensive biodistribution data for the prototype agent ¹²⁵I-NM324 inseveral tumor models has previously been reported. Tumor-to-blood ratiosexceeding 8:1 were seen at delayed times post-injection. For example, ina rat mammary tumor model, tumor-to-normal tissue ratios reached amaximum at 96 hours with a tumor-to-blood ratio of 8.6 andtumor-to-muscle ratio of 20:1. Moreover, the biodistribution ofPLE-associated radioactivity is heterogeneous in tumor, as demonstratedby microautoradiogram studies showing that the PLE radioactivity residesexclusively in viable tumor cells located toward the outer regionsrather than the central necrotic regions. Comparative biodistributiondata for NM324 and NM404 in SCID mice have been obtained in prostate andA549 lung cancer tumor models. These studies revealed hightumor-to-normal tissue ratios and tumor uptake exceeding 25% of theinjected dose with NM404, thus supporting the desire to examine thebiodistribution of PLE analogs in humans.

Isotope Selection

Because the tumor-targeting strategy of PLE analogs appears to involveselective tumor retention over time, relatively short-lived nuclidessuch as ¹⁸F or even ^(99m)Tc are not practical for labeling NM404 at thecurrent time. With a 13 h half-life and optimal imaging characteristics,iodine-123 may also prove suitable for this agent when scanned in a3-dimensional SPECT mode. Imaging with iodine-123 will require furtherinvestigation. While tumor localization of PLE analogs appears to occurwithin several hours of injection (NM324 images obtained 6 h postinjection in a lung cancer patient showed intense lung tumor uptake in aprior study), planar 2-dimensional imaging, like that currently beingperformed with iodine-131 in humans, requires a delay period to allowbackground activity to clear from neighboring normal tissues and blood.It is likely that earlier imaging may be possible when scanning with PETand 3D SPECT where neighboring radioactivity is less interfering due tothe 3-dimensional nature of these modalities. In organs where backgroundradioactivity remains inherently low (brain for example), it may bepossible to use gamma emitting isotopes like iodine-123 which inaddition to providing beautiful images, would permit image acquisitionlater on the same day of injection. Although the use of other isotopesmay ultimately prove suitable for use with NM404, the current focus willdevelop the PET imaging capability of this agent due to the recentsuccess of oncologic imaging using hybrid PET-CT scanners. Theunsurpassed diagnostic accuracy afforded by a biochemical or functionaltumor imaging PET agent combined with the precise anatomic accuracyprovided by CT is now the gold standard for tumor imaging. However, itis highly advantageous to label PLE analogs with iodine-124, arelatively new PET isotope, wherein the physical half-life (4 days)matches well with PLE tumor uptake and retention kinetics. LabelingNM404 with iodine-124 represents a natural extension of prior studieswith gamma-emitting nuclides. It has been shown that PET imaging with¹²⁴I affords over 40 times the sensitivity of planar ¹³¹I-gammascintigraphy. PET, unlike traditional gamma camera imaging, also offerssignificant resolution enhancement, image quantification, and3-dimensional capabilities. Due to the preliminary success of ¹³¹I-NM404in the current lung cancer imaging trial, it is now imperative to labelNM404 with iodine-124 and evaluate its tumor detection efficacy by PETin order to overcome the limitations inherently associated with planarscintigraphy

The utility of tumor tracers like ⁶⁷Ga-citrate and ¹⁸F-FDG is limited bytheir lack of specificity to distinguish neoplasm from inflammation.This lack of specificity is a significant clinical issue in patientswith cancer. However, preliminary studies with PLE analogs offerspromise in overcoming this limitation. Prior experiments conducted inrats revealed no uptake and retention of NM324 into carrageenan-inducedgranulomas. Gallium citrate, however, utilized as a control in thisstudy, did indeed concentrate significantly in the granulomatouslesions. Thus, this preliminary finding that PLE analogs don'tapparently localize in inflammatory lesions further justifies the needfor evaluation of this agent in human cancer patients. Although FDG-PEThas paved the way for hybrid imaging, its lack of tumor cell specificitywill always limit its diagnostic efficacy. New molecularly targetedagents, like NM404, which display universal tumor uptake and selectiveretention regardless of location, as well as selectivity for malignanttumor cells and not inflammatory or hyperplastic lesions, will representa significant improvement in the detection and characterization ofcancer.

While planar nuclear medicine imaging techniques have historicallyafforded acceptable 2D images, this modality offers no tomographiccapability and poor image quantitation. Although ¹²⁵I-NM404 was suitablefor preliminary scintigraphic imaging and tissue distribution studies inrodent tumor models and ¹³¹I was suitable for Phase 1 safety andpharmacokinetic evaluation in human lung cancer patients, neither areoptimal for quantitative in vivo human imaging. PET imaging withiodine-124, a relatively new and commercially available positron isotopewith a 4-day half-life, would alleviate many of the problems associatedwith planar imaging. Transitioning the unique tumor imaging capabilitiesof NM404 into PET scanning has now become a major goal of our lab sincethe iodine-124 became commercially available late last year. If thetumor specificity of NM404 for malignant tumors seen in mouse models isconfirmed in humans, then we would have an agent with more tumorselectivity than FDG, albeit without its inflammatory site localizationproperties. Moreover, the agent could be manufactured in one facilityand shipped to virtually any location in the world due to its 4-day halflife.

The inventors recently successfully radiolabeled NM404 twice withiodine-124 from a commercial vendor (Eastern Isotopes). Theradiochemical yield (>60% mean isolated yield, >99% purity) was verysimilar to what we usually obtain with commercial sources of iodine-125or 131 sodium iodide. The PET imaging characteristics of ¹²⁴I-NM404 in arat CNS-1 brain tumor model (FIG. 24) was investigated. Imaging times inthis preliminary study were limited to 24 h and 4 days post injectiondue to microPET scanner availability. MicroPET images acquired 24 hafter iv injection of ¹²⁴I-NM404 were corroborated withcontrast-enhanced MRI images and showed intense uptake of the tracer inthe brain tumor accompanied by little or no uptake in surrounding intactbrain tissue. This study represents the first PET image obtained withNM404 and demonstrates the ability to efficiently radiolabel, purify,and formulate NM404 for PET imaging. These compounds may then be usedfor extending NM404 PET utility into human cancer patients.

Determine Tumor Uptake and Retention Characteristics of ¹²⁴I NM404 byPET-CT in Patients with Radiographically Evident Metastatic ProstateCancer.

Objectives and Rationale

Assessment of whether selective detection of known metastatic lesions byradioiodinated NM404 is possible and comparable to conventionalradiologic modalities is discussed in the following paragraphs.Inclusion criteria for this study will consist of fifteen patients withmetastatic prostate cancer, with at least 5 patients having soft-tissueprostate cancer metastases and at least 5 of the patients with bonymetastases identifiable by conventional radiologic studies which includeCT scan and bone scan. Following patient enrollment, uptake ofradiolabeled NM404 will be measured by I-124 isotope PET-CT scan andcorrelated with the radiographically evident lesions detected by thepatient's other conventional staging studies.

Methods

Synthesis, Radiolabeling, and Formulation:

Radioiodination of stable NM404 with ¹²⁴I-sodium iodide is routinelyachieved by modification of an ammonium sulfate-mediated isotopeexchange reaction reported by Mangner and recently optimized for NM404in our lab. Exchange reaction methodology has been used effectively forinitial human trials with NM324, the predecessor of NM404 and iscurrently being used for preclinical studies and the human lung cancertrial. Briefly, a 2-ml glass vial is charged with 10 mg of ammoniumsulfate dissolved in 50 μl of deionized water. Fourteen 2 mm chemicalresistant glass beads are added, a Teflon lined septum and screw cap areadded and the vial gently swirled. A solution of 20 μg (in 20 μl ofethanol) of stock NM404 is added followed by aqueous sodium iodide (124,1-5 mCi) in less than 30 μl aqueous 0.01 N sodium hydroxide. The isotopesyringe is rinsed with three 20-μl portions of ethanol. The reactionvile is swirled gently. A 5-ml disposable syringe containing glass woolin tandem with another 5-ml charcoal nugget filled syringe with needleoutlet are attached. The glass wool syringe acts as a condensationchamber to catch evaporating solvents and the charcoal syringe trapsfree iodide/iodine. The reaction vessel is heated in a heating blockapparatus for 45 minutes at 150° C. Four 20 ml volumes of air areinjected into the reaction vial with a 25-ml disposable syringe andallowed to vent through the dual trap attachment. The temperature israised to 160° C. and the reaction vial is heated another 30 minutes.After cooling to room temperature, ethanol (200 μl) is added and thevial swirled. The ethanolic solution is passed through apre-equilibrated Amberlite IRA 400-OH resin column to remove unreactediodide. The eluent volume is reduced to 50 μl via a nitrogen stream (usecharcoal syringe trap) and the remaining volume injected onto an HPLCsilica gel column (Perkin Elmer, 3 μm×3 cm disposable cartridge columneluted at 1 ml/min with hexane/isopropanol/water (52:40:8)) forpurification. Final purity is determined by TLC (plastic backed silicagel-60 eluted with chloroform-methanol-water (65:35:4, Rf=0.1). The HPLCsolvents are removed by rotary evaporation and the resultingradioiodinated NM404 is solubilized in aqueous 2% pharmaceutical gradePolysorbate-20 (0.1 μl/mg of compound). The ethanol is removed undervacuum and the residue dissolved in sterile water to give a finalsolution containing no more than 2-3% Polysorbate-20. Sterilization willbe achieved by filtration through a sterile 0.2 μm filter unit. Each ofthe solutions will be tested for pyrogens using the Limulus AmebocyteLysate test kit. This is the same procedure currently employed for thepreparation, purification and sterile formulation of I-131-labeled NM404for lung cancer patient studies. The drug Master Formulation Card andProduct Preparation Checklist are included in the supplemental sectionof this proposal. We have performed this radioiodination hundreds oftimes with either iodine-131 or 125 and typically achieve radiochemicalyields ranging from 60-90% (isolated pure product) with specificactivities of exceeding 130 mCi/μmol of NM404. This method has affordedsufficient pure injectable ¹³¹I-NM404 for our ongoing preclinical andhuman trials. We have not had a labeling failure to date. Moreover, andrelevant to the current proposal, we have recently successfullyradiolabeled NM404 with iodine-124 sodium iodide (5 mCi) from EasternIsotopes in >60% isolated radiochemical yield. Although acceptancecriteria require >95% radiochemical purity, results typically exceed 99%since the entire reaction mixture is subjected to preparative HPLCpurification.

I-124 Dose for Human Administration:

The toxicity profile, biodistribution, kinetics, optimal imaging times,and dosimetry of ¹³¹I NM404 are currently being evaluated in an ongoingstudy in patients with lung cancer (NSCLC) at UWCCC, as discussed above.Dosing of radioiodinated ¹²⁴I NM404 will be based on MIRD extrapolationof the iodine-131 dosimetry data derived from this ongoing study. The¹³¹I-NM404 dose is currently calculated as follows: Animalbiodistribution data is used to determine the percentage of injecteddose/organ at varying time points. These animal data are extrapolated toman by means of MIRD formalism (MIRDOSE PC v3.1) using standardconversion factors for differences in organ mass and anatomy between ratand standard man, providing predicted human organ doses; Based on thesepredicted doses, the permissible mCi dose to be injected into humans isdetermined using the maximal doses legally permitted by RDRC regulationsfor specific human tissue as defined in the Federal Register (21CFR Part361.1) e.g. whole body, blood, blood forming tissues, eye lens, gonads—3rem/dose; any other tissue—5 rem/dose. This approach was previously usedto bring both NM324 and NM404 to clinical use, resulting in a calculateddose of 1 mCi. New animal data acquired with NM404 (5 rad testesdose=2.1 mCi and 5 rad dose to adrenals=2.2 mCi) indicate the startingmaximum dose of ¹³¹I-NM404 will be around 1.9 mCi or twice that ofNM324.

Study Procedures:

Before infusion, an intravenous line will be established in the arm. Thetracer dose <0.3 μg/kg body weight ¹²⁴I-NM404 (1 mCi, actual dosepending current study dosimetry results) will be infused over 5 minutes.The preparation will be sterile, pyrogen-free, and contain <5% freeiodine by thin layer chromatography (usual result is <1%). The patientwill be clinically monitored for adverse reactions during the infusion.

Since the animal toxicity studies (using over 1,000 times the maximumplanned quantity of ¹²⁴I-NM404 in this study) and the preliminaryclinical safety studies with the unlabeled compound (at doses 10 timesgreater) were without side effects, we do not anticipate toxicity at theimaging dose. However, we are cognizant of the possibility of untowardallergic or other reactions. The patients' vital signs (pulse, BP,temperature and respiratory rate) will be re-checked immediately afterthe infusion and at 30 and 60 minutes post injection. The vital signswill then be checked each hour for two hours.

Baseline PET-CT images (whole body and selected regional conjugateviews) for the first five patients will be obtained at 48 and 96 hourspost injection. PET images will be obtained using a GE Clinical GEDiscovery LS PET-CT scanner utilizing appropriate attenuation correctionand optimized to iodine-124. The 48 and 96 hour time points are based onpreliminary data regarding optimal imaging times for ¹³¹I-NM404 in lungcancer, however, the optimum tumor-targeting time for ¹²⁴I-NM404 inhuman prostate cancer is yet to be determined.

¹²⁴I-NM404 tracer accumulation and washout will be recorded on the GEDiscovery LS PET/CT scanner or on GE Advance PET scanner available atour clinic. A CT scan will be performed first for patient localizationand attenuation correction on the Discovery LS PET/CT scanner; atransmission scan will be performed on the Advance PET scanner. Each PETscan will be corrected for random, dead time, attenuation, and scatter.First scanning will be performed immediately at 48 and 96 h after the¹²⁴I-NM404 injection. Whole body scans will be performed with increasedscanning time over the tumor site (up to 30 min). Images will bereconstructed using an iterative OSEM reconstruction method withattenuation correction, and smoothed with a Gaussian filter.

Data evaluation will be based on a region-of-interest (ROI) analysis ofcoregistered PET images. CTs from each PET/CT scan will be co-registeredto enable accurate positioning of the PET data. ROIs will be outlined torepresent various organs in the field of view (typically heart, lung,muscle, liver, stomach, spleen, intestine, kidney, and bladder). Foreach ROI and for each time frame, the average radioactivityconcentration will be calculated and standardized uptake values (SUVs)calculated. Detailed pharmacokinetic analysis will not be performed inthis study, but will have been completed in the ongoing lung cancertrial and not duplicated here, because this will be performed in thelung cancer trial, wherein 2 patients will undergo PET scanningimmediately after, and subsequently at 6, 24, and 48 h post NM404infusion. SUVs will be plotted as time-activity curves. The time courseof radioactivity in each ROI will be fitted using various kinetic modelsincluding a two-rate constant in a two compartment model and a four-rateand three-rate constant, in a three compartment model in conjunctionwith the non-linear regression fitting. The nonlinear least-squarefitting will be done using a modified gradient-expansion algorithm. Thebest fits will be determined by minimizing a χ² function with respect tovariations in the model parameters. For the 3 k model the data will befit with k₄ set equal to 0 in order to test the assumption of thephospholipid irreversible trapping in the tumor membrane. In addition,Patlak plots will be used to standardize the kinetics of tissueradioactivity to the kinetics of plasma radioactivity.

Statistical Considerations:

This is a feasibility study to determine the tumor uptake and retentioncharacteristics of radioiodinated ¹²⁴I NM404 by PET-CT in patients withclinically evident metastatic prostate canter. Fifteen patients withmetastatic prostate cancer will be accrued for Part 1 of this study.Image scans will be scored using a typical 0-3 scale: 0=no perceptibleuptake, 1+=uptake barely perceptible above background, 2+=uptake clearlydistinguishable above background, 3+=intense uptake, much greater thansurrounding normal structures (2+ and 3+ are considered abnormal orpositive for lesion identification; 0 and 1+ are considered normal ornegative for lesion identification). An uptake rate classified aspositive for lesion identification of at most 50% would be considered asclinically irrelevant. Therefore, we will test the null hypothesis thatthe uptake rate classified as positive for lesion identification will beat most 50% versus the alternative hypothesis that the rate is greaterthan 50%. We anticipate the uptake rate classified as positive forlesion identification will be at least 80%. Assuming a total sample sizeof 15 patients, the one-sample binomial test with the null hypothesisthat the uptake rate classified as positive is at most 50% has 85% powerto detect a rate of 80% at the (one-sided) 10% significance level. Arate of 90% will be detected with 95% power. With a sample size of 15patients, the proportion of tumors classified as positive for lesionidentification will have a standard error of at most 13% and the 90%confidence interval for the proportion will be no wider than 39%.

Determine the Specific Tumor Accumulation and Metabolic Fate of ¹²⁴INM404 in Patients with Clinically Organ-Confined Prostate Cancer Who areCandidates for a Radical Prostatectomy with Bilateral Pelvic Lymph NodeDissection.

Objectives and Rationale

As a secondary exploratory analysis, malignant and benign prostatic andnodal tissue obtained by radical prostatectomy and bilateral lymph nodedissection will be evaluated for radioiodinated NM404 accumulation andmetabolites of NM404 and data from this tissue analysis will be comparedwith the results of final pathology analysis. Ten patients withorgan-confined prostate cancer who are scheduled to undergo a radicalprostatectomy with bilateral pelvic lymph node dissection will beenrolled. All patients will undergo conventional preoperative stagingstudies which include CT scan and bone scan. All patients will also havean NM404 PET-CT scan the week before the scheduled prostatectomy. Finalpathologic analysis of the resected prostate and lymph nodes will becorrelated with results of the NM404 accumulation and metabolite tissueassay to determine relationship of signal to tumor volume in the primaryspecimen and lymph nodes in patients with locoregional metastaticdisease. Nodal metastases detected by the NM404 accumulation despite anegative preoperative CT scan and bone scan would have great clinicalsignificance in terms of improved staging of these patients prior totreatment.

Study Procedures:

Imaging procedures in this study will be identical to the first study,except that patients will be imaged only once, based upon the optimalimaging time as identified in the first protocol (probably between 24-48hours after the injection). Six prostate biopsy cores will be obtainedin sextant distribution (right and left apex, mid-gland and baseregions) from the prostate specimen in the OR immediately followingsurgical removal. These six biopsy cores will be evaluated by frozensection pathology to ensure a representative sampling of both malignantand benign prostate tissue. A second set of 6 biopsy cores obtainedsimultaneously from the same corresponding locations in the prostatewill be analyzed for uptake of ¹²⁴I-NM404. Accordingly, they will bephotographed and undergo high-resolution scanning on a Bioscan AR2000radioscanner and also weighed and radioactivity quantitated in a wellcounter. Radioactivity concentration will be determined on a % dose/gtissue sample basis for comparison. These results will be compared tohistology results in order to confirm localization in tumor relative tosurrounding uninvolved tissue. The core biopsies will be labeled withthe patient's study identification number and location in the prostatefrom which it was obtained.

Final pathology of any lymph nodes harboring metastatic prostate tumorwill also be correlated with ¹²⁴I-NM404 uptake signal detected by thepreoperative PET-CT scan. Vital signs will be obtained post ¹²⁴I-NM404infusion.

Statistical Considerations:

en patients with high-risk clinically localized prostate cancer will beaccrued for Part 2 of the study. The null hypothesis for Part 2 is thatthe log ratio of the accumulation of NM404 in tumor tissues to that innormal surrounding tissues in clinically localized prostate cancerpatients is zero. The alternative hypothesis is that the log ratio ofthe accumulation of NM404 is greater than 0. A tumor-to-adjacent normaltissue (T/N) ratio of at least 5:1 would be considered as clinicallyrelevant. Based on past experience with NM404, it is anticipated thatthe standard deviation of the log ratio of the accumulation of NM404 isbetween 1.5 and 2.0. With a sample size of 10, the one-sided t-test witha one-sided 10% significance level has 86%, 90% and 95% power to detectthe effect size of 0.8, 0.90, and 1.00, respectively. For example, ifthe standard deviation of the log ratio is 2.0, the 5:1 ratio in theaccumulation of NM404 in the tumor to normal tissues will be detectedwith 86% power. Analogously, if the standard deviation of the log ratiois 1.6, a ratio of 5:1 in the accumulation of NM404 in tumor to normaltissues will be detected with 95% power.

Determine Whether NM404 can Detect Metastatic Recurrence FollowingPrimary Treatment of Prostate Cancer in Ten Patients with a Rising PSAas their Only Evidence of Disease (Stage D0).

Objectives and Rationale

By definition, patients with stage D0 prostate cancer have a rising PSAfollowing definitive treatment of their disease, signifying biochemicalrecurrence, with conventional staging studies including CT scan and bonescan which are negative for radiographic evidence of disease. Patientsmore likely to have disease detectable by the NM404 PET-CT scan will beenrolled such that they must have a PSA rise of greater than 0.75ng/ml/year.

Study Procedures:

Imaging procedures in this study will be identical to the first study,except that patients will be imaged only once, based upon the optimalimaging time as identified in the first protocol. Patients will receivean injection of ¹²⁴I-NM404 (0.3 μg/kg body weight, 1 mCi or the limitestablished by dosimetry calculations in the first set of patients).Patients will also have a PET-CT scan 48 hours after infusion. Vitalsigns will be obtained post ¹²⁴I-NM404 infusion.

Statistical Considerations:

Ten patients with stage D0 prostate cancer will be accrued andfollowed-up for metastatic recurrence for at least 2 years. Since allpatients accrued in this part of the study will have PSA values greaterthan 0.75 ng/ml/year, we expect that at least 60% will experiencemetastatic recurrence within 2 years. Assuming that 6 out of the 10patients will eventually experience metastatic recurrence within 2years, the sensitivity of NM404 to detect metastatic recurrence will beestimated with a standard error of less than 20% and the 90% confidenceinterval for the sensitivity will be no wider than 55%. Furthermore, thenull hypothesis that the sensitivity of NM404 in this patient populationis at most 30% will be tested against the alternative that thesensitivity is greater than 30%. The one-sample binomial test has 83%power to detect a sensitivity of 75% at the one-sided 10% significancelevel. A sensitivity of 80% will be detected with 90% power.

Approximately 230,110 new cases of prostate cancer will be diagnosed inthe United States for the year 2004 alone. Despite technical refinementsin definitive local treatment of clinically organ confined prostatecancer by radical prostatectomy, such that many men are cured withprimary therapy alone, as many as 40% of patients will experiencebiochemical recurrence with long-term follow-up. One of the greatestchallenges in treating patients with clinically organ confined prostatecancer or patients with biochemical recurrence following definitivetreatment of presumed organ-confined disease remains to accuratelydistinguish localized versus metastatic disease. This diagnosticcapability is important to identify patients who may benefit fromeffective local treatment modalities including surgery, external beamradiation, brachytherapy, and cryotherapy. Because we presently do nothave an accurate means of staging, patients with occult metastaticdisease may unnecessarily undergo local treatment with associated risksof therapy. Furthermore, patients with a rising PSA due to localrecurrence, in whom systemic recurrence cannot be excluded withconfidence, may unnecessarily undergo hormonal ablation, which isgenerally not considered curative and is associated with osteoporosisdevelopment, decreased libido, weight gain, menopausal symptoms, andoverall malaise, as well as the evolution of hormonally independentprostate cancer.

While conventional imaging studies such as computed tomography (CT) andmagnetic resonance imaging (MRI) are useful in assessing soft-tissuemetastasis, the vast majority of prostate cancer metastasizes to thebone only. Thus, the utility of CT and MRI scanning in assessing thedisease is suboptimal and more sensitive imaging modalities for eitherlocally recurrent or metastatic prostate cancer are necessary.Radioimmunoscintigraphy with Indium-111 capromab pendetide (ProstaScint,Cytogen Corp, Princeton, N.J.) has been utilized in patients followingprostatectomy with a rising PSA who have a high clinical suspicion ofoccult metastatic disease and no clear evidence for metastatic diseasein other imaging studies. Use of the ProstaScint scan for patients atrisk for occult metastases from prostate cancer remains controversialhowever. One approach to the development of a sensitive imaging exam isto develop a more appropriate carrier molecule, which is key toachieving delivery of a radiopharmaceutical probe to the desired targettissue. Our strategy has been to study radioiodinated phospholipid etheranalogs (PLE) as potential diagnostic imaging agents, to capitalize onthe unique biochemical or pharmacological properties of these moleculesresulting in a high degree of tissue or tumor selectivity. Inpreclinical models, we have shown these molecules selectively accumulatein a wide variety of murine and human tumors in high levels.

Certain embodiments of the present invention provides preliminary dataregarding the use of the second-generation PLE analog, NM404, in imagingpatients with prostate cancer. It has been shown that NM404 is (a)selectively retained in a wide variety of tumor types in preclinicalmodels, with a high degree of sensitivity, (b) is safe in humans, (c)can be radiolabeled with I-124, and (d) has appropriate dosimetrycharacteristics labeled with I-131.

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All references cited herein are specifically incorporated by referencein their entireties and for all purposes as if fully set forth herein.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the following claims.

1-20. (canceled)
 21. A method, comprising: determining cancer in asubject to which ¹²⁴I-labeled 18-(p-iodophenyl)octadecyl phosphocholinehas been administered by determining ¹²⁴I radioactivity in the subject.22. The method of claim 21, wherein the subject is human.
 23. The methodof claim 21, comprising determining ¹²⁴I-radioactivity using PETscanning.
 24. The method of claim 21, comprising determining¹²⁴I-radioactivity using SPECT scanning.
 25. The method of claim 21,comprising determining ¹²⁴I-radioactivity using gamma camerascintigraphy.
 26. The method of claim 21, comprising determining¹²⁴I-radioactivity using MRI.
 27. The method of claim 21, comprisingadministering between about 0.5 μCi and about 500 mCi of the¹²⁴I-labeled 18-(p-iodophenyl)octadecyl phosphocholine to the subject.28. The method of claim 21, comprising administering less than 0.3 μg/kgof body weight of the ¹²⁴I-labeled 18-(p-iodophenyl)octadecylphosphocholine to the subject.
 29. The method of claim 21, wherein thecancer is lung cancer.
 30. The method of claim 21, wherein the cancer isadrenal cancer.
 31. The method of claim 21, wherein the cancer ismelanoma or subcutaneous cancer.
 32. The method of claim 21, wherein thecancer is intestinal cancer, colon cancer or colorectal cancer.
 33. Themethod of claim 21, wherein the cancer is ovarian cancer or cervicalcancer.
 34. The method of claim 21, wherein the cancer is prostatecancer.
 35. The method of claim 21, wherein the cancer is liver canceror hepatocellular carcinoma.
 36. The method of claim 21, wherein thecancer is squamous cell carcinoma.
 37. The method of claim 21, whereinthe cancer is retinoblastoma.
 38. The method of claim 21, wherein thecancer is glioma.
 39. The method of claim 21, wherein the cancer isbreast cancer.
 40. The method of claim 21, wherein the cancer iscarcinosarcoma.
 41. The method of claim 21, wherein the cancer ispancreatic cancer.
 42. A method of differentiating inflammation,adenoma, hyperplasia, or neoplasia in a subject, the method comprising:providing a subject to which ¹²⁴I-labeled 18-(p-iodophenyl)octadecylphosphocholine has been administered; and determining whether thesubject has inflammation, adenoma, hyperplasia, or neoplasia bydetermining ¹²⁴I radioactivity in the subject.
 43. The method of claim42, comprising determining ¹²⁴I-radioactivity using PET scanning. 44.The method of claim 42, comprising determining ¹²⁴I-radioactivity usingSPECT scanning.
 45. The method of claim 42, comprising determining¹²⁴I-radioactivity using gamma camera scintigraphy.
 46. The method ofclaim 42, comprising determining ¹²⁴I-radioactivity using MRI.
 47. Amethod for determining radiation- and chemo-insensitive cancer or cancermetastasis, comprising: providing a subject to which ¹²⁴I-labeled18-(p-iodophenyl)octadecyl phosphocholine has been administered; anddetermining whether the subject has radiation- and chemo-insensitivecancer or cancer metastasis by determining ¹²⁴I radioactivity in thesubject.
 48. The method of claim 48, comprising determining¹²⁴I-radioactivity using PET scanning.
 49. The method of claim 48,comprising determining ¹²⁴I-radioactivity using SPECT scanning.
 50. Themethod of claim 48, comprising determining ¹²⁴I-radioactivity usinggamma camera scintigraphy.
 51. The method of claim 48, comprisingdetermining ¹²⁴I-radioactivity using MRI.